WO2018141089A1 - 广谱抑制hiv的脂肽、其衍生物、其药物组合物及其用途 - Google Patents

广谱抑制hiv的脂肽、其衍生物、其药物组合物及其用途 Download PDF

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WO2018141089A1
WO2018141089A1 PCT/CN2017/072885 CN2017072885W WO2018141089A1 WO 2018141089 A1 WO2018141089 A1 WO 2018141089A1 CN 2017072885 W CN2017072885 W CN 2017072885W WO 2018141089 A1 WO2018141089 A1 WO 2018141089A1
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polypeptide
hiv
sequence
derivative
amino acid
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PCT/CN2017/072885
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English (en)
French (fr)
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何玉先
种辉辉
丁晓慧
熊胜文
朱园美
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中国医学科学院病原生物学研究所
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Priority to PCT/CN2017/072885 priority Critical patent/WO2018141089A1/zh
Publication of WO2018141089A1 publication Critical patent/WO2018141089A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a lipopeptide, a derivative thereof, a pharmaceutical composition thereof and a use thereof for broad spectrum inhibition of HIV in the field of biomedicine.
  • HIV Human immunodeficiency virus
  • type 1 Human immunodeficiency virus
  • type 2 Human immunodeficiency virus
  • HIV-1 is the main pathogen, and about 1 million to 2 million people are caused by HIV-2 infection, mostly in West Africa.
  • HIV-2 has gradually spread to Europe, Asia and North America, posing a serious threat to human health [1].
  • Vaccines are the best way to prevent AIDS, but effective AIDS vaccines are unlikely to have major breakthroughs in the near future. Therefore, the development of drugs that block different stages of replication of the virus is currently the key strategy for prevention and control of AIDS. Because the current clinical application of AIDS treatments are based on HIV-1 design, many drugs have limited activity on HIV-2 and cannot be used for clinical treatment of HIV-2 infection.
  • Env envelope glycoprotein
  • the protein is formed by a non-covalent bond between the surface subunit gp120 and the transmembrane subunit gp41, and has a trimer structure in a natural state.
  • the main function of gp120 is to bind to the cellular receptor CD4 and co-receptors (such as the chemokine receptor CCR5 or CXCR4, etc.), while gp41 mainly mediates membrane fusion of viruses and cells.
  • FP hydrophobic fusion peptide
  • NHR N-terminal helical repeat
  • CHR C-terminal helical repeat
  • the core structure of gp41 is a six-helix bundle (6-HB), and three NHRs consist of N-
  • the helix interacts with the amino acid residues at the a and d positions to form a centrally located helical trimer with amino acid residues at the e and g positions exposed to the periphery of the centrosome and with a C-helix composed of three CHRs.
  • the a and d positions interact [3, 4].
  • the C-helices are respectively combined in the grooves formed by the three N-helices in an anti-parallel manner.
  • T-20 Enfuvirtide, Fuzeon
  • an AIDS-approved drug approved by the US FDA in 2003 is a 36-amino acid-derived polypeptide derived from CHR.
  • T-20 is the first and currently the only viral membrane fusion inhibitor approved for clinical treatment, but it is easy to induce drug resistance, and it is HIV-2 Poor activity greatly limits its wide application [9].
  • T1249 is the second generation of T20 with high ability to inhibit HIV-1 and HIV-2, but clinical trials have been discontinued due to its dosage form and cost. Therefore, many studies have focused on the development of a new generation of HIV membrane fusion inhibitory peptides. Since T-20 does not contain an important NHR pocket binding sequence (PBD), another CHR polypeptide C34 is widely used as a design template for novel inhibitors [7]. However, most newly designed peptides have the same long amino acid sequence as T-20 and T1249, such as T2635 containing 38 amino acids and SC35EK containing 35 amino acids. Sifuvirtide (SFT) and ibbevirin (ABT) in Chinese clinical trials have 36 and 34 amino acids, respectively.
  • SFT ifuvirtide
  • ABT ibbevirin
  • One technical problem to be solved by the present invention is how to increase the antiviral activity of a polypeptide having HIV-1 and/or HIV-2 and/or SIV activity against HIV-1 and/or HIV-2 and/or SIV, and/or Extend the duration of its antiviral activity.
  • the present invention provides a lipopeptide having anti-HIV-1 and/or HIV-2 and/or SIV activity.
  • the lipopeptide (compound) having anti-HIV-1 and/or HIV-2 and/or SIV activity provided by the present invention is as follows a) or b):
  • the lipopeptide comprises a polypeptide having antiviral activity, a tether attached to the carboxy terminus of the polypeptide, an amino acid X residue linked to the tether and a lipophilic compound linked to the amino acid X residue.
  • the amino acid X is K, C, S, T or Y;
  • the lipopeptide comprises a polypeptide having antiviral activity, a lipophilic compound linked to the carboxy terminus of the polypeptide;
  • the virus is any one of the following v1-v7:
  • V1 HIV-1, HIV-2 and SIV;
  • the antiviral activity may also be referred to as inhibiting viral activity, specifically inhibiting virus for cell fusion and/or inhibiting virus invading cells. And / or inhibit viral replication.
  • the linking arm may be a flexible linking arm such as Fmoc-NH-PEGn-CH 2 CH 2 COOH, n is 2, 3, 4, 6, 8, 10, 11 or 12, and the connecting arms can also be replaced by other flexible connecting arms known in the art; and/or,
  • the lipophilic compound may be a fatty acid having 8 to 20 carbon atoms or a cholesterol (cholesterol) or a dihydrosphingosine; the lipophilic compound may be a tocopherol. Wait.
  • the fatty acid having 8 to 20 carbon atoms may be palmitic acid (also known as palmitic acid) (C16) or stearin. Acid (C18).
  • all of the amino acids in the polypeptide having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV may be L-form.
  • the D-type amino acid refers to an amino acid corresponding to the L-form amino acid constituting the protein
  • the artificially modified amino acid refers to a common L-type amino acid which constitutes a protein modified by methylation, phosphorylation, etc.
  • the rare amino acid existing in nature includes a constituent protein Uncommon amino acids and amino acids that do not constitute proteins, such as 5-hydroxylysine, methylhistidine, gamma aminobutyric acid, homoserine, and the like.
  • the polypeptide is any one of P1-P20:
  • P1 a polypeptide represented by SEQ ID NO: 1 in the sequence listing; that is, a polypeptide represented by amino acid residues 1 to 23 of LP-19, LP-20, LP-21, LP-22 and LP-23 in Fig. 1;
  • P3 a polypeptide represented by SEQ ID NO: 2 in the sequence listing; that is, a polypeptide represented by amino acid residues 1-31 of LP-31 in Fig. 1;
  • P4 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 2 in the sequence table, the derived polypeptide having the antiviral activity;
  • P5 the polypeptide shown in SEQ ID NO: 3 in the sequence listing; that is, amino acid residues 1-31 of LP-32 in Fig. 1 a polypeptide represented by a base;
  • P6 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 3 in the sequence table, the derived polypeptide having the antiviral activity;
  • P7 a polypeptide represented by SEQ ID NO: 4 in the sequence listing; that is, a polypeptide represented by amino acid residues 1-34 of LP-29 in Fig. 1;
  • P8 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 4 in the sequence table, the derived polypeptide having the antiviral activity;
  • P9 a polypeptide represented by SEQ ID NO: 5 in the sequence listing; that is, a polypeptide represented by amino acid residues 1 to 25 of LP-25 in Fig. 1;
  • P10 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 5 in the sequence table, the derived polypeptide having the antiviral activity;
  • P11 a polypeptide represented by SEQ ID NO: 6 in the sequence listing; that is, a polypeptide represented by amino acid residues 1 to 25 of LP-24 in Fig. 1;
  • P12 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 6 in the sequence table, the derived polypeptide having the antiviral activity;
  • P13 a polypeptide represented by SEQ ID NO: 7 in the sequence listing; that is, a polypeptide represented by amino acid residues 1-31 of LP-28 in Fig. 1;
  • P14 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 7 in the sequence table, the derived polypeptide having the antiviral activity;
  • P15 a polypeptide represented by SEQ ID NO: 8 in the sequence listing; that is, a polypeptide represented by amino acid residues 1-30 of LP-30 in Fig. 1;
  • P16 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 8 in the sequence table, the derived polypeptide having the antiviral activity;
  • P17 a polypeptide represented by SEQ ID NO: 9 in the sequence listing; that is, a polypeptide represented by amino acid residues 1-27 of LP-26 in Fig. 1;
  • P18 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide represented by the sequence 9 in the sequence table, the derived polypeptide having the antiviral activity;
  • P19 a polypeptide represented by SEQ ID NO: 10 in the sequence listing; that is, a polypeptide represented by amino acid residues 1-28 of LP-27 in Fig. 1;
  • P20 a derivative polypeptide obtained by adding or substituting one or more amino acid residues at any position of the polypeptide of SEQ ID NO: 10 in the Sequence Listing, said derived polypeptide having said antiviral activity.
  • the derivative polypeptide consists of 20 to 34 amino acid residues.
  • the lipopeptide having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV, in the a) or b), in order to improve stability, the lipopeptide further includes an amino terminal protecting group and/or a carboxyl group.
  • the lipopeptide having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV, the lipopeptide is af) or bf):
  • Af a polypeptide having antiviral activity, a tether attached to the carboxy terminus of the polypeptide, an amino acid X residue linked to the tether, and a lipophilic compound linked to the amino acid X residue a protecting group composition
  • the amino acid X is K, C, S, T or Y
  • the protecting group is an amino terminal protecting group and/or a carboxy terminal protecting group, and the amino terminal protecting group is attached to the amino group of the lipopeptide a carboxy terminal protecting group attached to the carboxy terminus of the lipopeptide;
  • the polypeptide having antiviral activity consisting of the polypeptide having antiviral activity, a lipophilic compound and a protecting group linked to the carboxy terminus of the polypeptide;
  • the protecting group is an amino terminal protecting group and/or a carboxy terminal protecting group, the amino group A terminal protecting group is attached to the amino terminus of the lipopeptide, and the carboxy terminal protecting group is attached to the carboxy terminus of the lipopeptide.
  • the amino terminal protecting group of the present invention may be any one of an acetyl group, an amino group, a maleoyl group, a succinyl group, a t-butoxycarbonyl group or a benzyloxy group or another hydrophobic group or a macromolecular carrier group;
  • the protecting group can be any of an amino, carboxyl, amido or t-butoxycarbonyl group or other hydrophobic group or macromolecular carrier group.
  • the tether is Fmoc-NH-PEG8-CH 2 CH 2 COOH.
  • Fmoc-NH-PEG8-CH 2 CH 2 COOH The English name for Fmoc-NH-PEG8-CH 2 CH 2 COOH is 1-(9H-fluoren-9-ylmethyl)ester or 5,8,11,14,17,20,23,26-Octaoxa-2-azanonacosanedioic acid , C 34 H 49 NO 12) .
  • Fmoc-NH-PEG8-CH 2 CH 2 COOH can be composed of two Fmoc-NH-PEG4-CH 2 CH 2 COOH (English name is Fmoc-15-amino-4, 7, 10, 13-tetraoxapentadecacanoic acid) is made in series.
  • the above lipopeptide having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV may specifically be any of the 14 lipopeptides of LP-19 to LP-32 in Fig. 1.
  • polypeptide of any of the above P1 to P20, a pharmaceutically acceptable salt thereof, or a derivative thereof is also within the scope of the present invention.
  • the derivative of the polypeptide may specifically be at least one of the following 1) to 5):
  • Multimers of PM1 or PM2 are also within the scope of the invention:
  • PM1 a multimer formed from the lipopeptide, a pharmaceutically acceptable salt thereof, or a derivative thereof;
  • compositions are also within the scope of the invention: a composition comprising C1) and C2): C1) is C11), C12) or/and C13); said C11) is said lipopeptide, a derivative thereof, or a pharmaceutically acceptable salt thereof; said C12) is said polypeptide, a derivative thereof, or a pharmaceutically acceptable salt; said C13) is said multimer;
  • composition has at least one of the following F1)-F5):
  • the virus is any one of the following v1-v7:
  • V1 HIV-1, HIV-2 and SIV;
  • the E1) is an antiviral product such as a drug or a vaccine
  • the E2) is a product, such as a drug or a vaccine, for treating and/or preventing and/or adjuvant treatment of a disease caused by a viral infection, such as AIDS;
  • the E3) is a product for inhibiting virus cell fusion, such as a drug or a vaccine
  • the E4) is a product that inhibits virus invading cells, such as a drug or a vaccine;
  • the E5) is a product that inhibits viral replication, such as a drug or a vaccine
  • the virus is any one of the following v1-v7:
  • V1 HIV-1, HIV-2 and SIV;
  • the present invention provides a pharmaceutical compound.
  • the pharmaceutical compound provided by the present invention is the C11), the C12) or the C13).
  • the pharmaceutical compound has at least one of the following F1) to F5):
  • the virus is any one of the following v1-v7:
  • V1 HIV-1, HIV-2 and SIV;
  • a method of treating or/and preventing a virus-infected animal comprising administering to the recipient animal the C11), the C12), the C13) or/and C14) to inhibit viral infection of the animal;
  • the virus is any one of the following v1-v7:
  • V1 HIV-1, HIV-2 and SIV;
  • the lipopeptide or polypeptide, derivative thereof, or pharmaceutically acceptable salt thereof provided by the present invention, the polymer, the composition or the pharmaceutical compound can be used for HIV (HIV-1 and/or HIV) -2) and/or treatment and/or prevention of SIV infection.
  • HIV HIV-1 and/or HIV
  • the lipopeptide or polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, the multimer, the composition or the pharmaceutical compound can be directly administered to a patient as a medicament, or The appropriate carrier or excipient is mixed and administered to the patient for the purpose of treating and/or preventing HIV infection.
  • the carrier materials herein include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers. Materials (such as cellulose acetate phthalate and carboxymethylcellulose, etc.). Among them, preferred are water-soluble carrier materials. These materials can be used in a variety of dosage forms including, but not limited to, tablets, capsules, pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Oral tablets, suppositories, freeze-dried powder injections, etc.
  • water-soluble carrier materials such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.
  • poorly soluble carrier materials such as ethyl cellulose, cholesterol stearate, etc.
  • enteric carriers such as cellulose acetate
  • ⁇ may be a general preparation, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems.
  • various carriers well known in the art can be widely used.
  • carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid.
  • wetting agent and binder such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin paste, sodium carboxymethyl cellulose , shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc.
  • disintegrating agents such as dried starch, alginates, agar powder, brown algae starch, sodium bicarbonate and tannic acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.
  • absorption promotion Agents such as quaternary ammonium salts, sodium lauryl sulfate, and the like
  • lubricants such as talc, silica,
  • Tablets may also be further formed into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets.
  • various carriers well known in the art can be widely used.
  • the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin , ethanol, honey, liquid sugar, rice paste or batter; etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, and the like.
  • a unit dosage form as a suppository various carriers well known in the art can be widely used.
  • the carrier are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.
  • an injectable preparation such as a solution, an emulsion, a lyophilized powder, and a suspension
  • all diluents conventionally used in the art for example, water, ethanol, polyethylene glycol, 1, may be used.
  • an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a conventional solubilizer, a buffer, a pH adjuster or the like may be added.
  • coloring agents, preservatives, perfumes, flavoring agents, sweeteners or other materials may also be added to the pharmaceutical preparations as needed.
  • the above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, and intraluminal injection; intraluminal administration, such as transrectal and vaginal; respiratory administration, such as transnasal; mucosal administration.
  • the above administration route is preferably administered by injection.
  • the lipopeptide or polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, the multidrug, the composition or the pharmaceutical compound to be administered depends on a number of factors, such as prevention or treatment of the disease The nature and severity of the patient, the gender, age, weight and individual response of the patient, the specific active ingredient used, the route of administration and the number of doses administered.
  • the above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.
  • the lipopeptide or polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, the multimer, the composition or the pharmaceutical compound can be directly used for the treatment and prevention of HIV-infected persons, or Used in combination with one or more anti-HIV drugs to achieve an overall therapeutic effect.
  • anti-HIV drugs include, but are not limited to, reverse transcriptase inhibitors, protease inhibitors, invasion inhibitors, integration inhibitors, and maturation inhibitors.
  • the above reverse transcriptase inhibitors may be AZT, 3TC, ddI, d4T, ddT, TDF, Abacavir, Nevirapine, One or more of Efavirenz and Delavirdine;
  • the protease inhibitor may be one or more of Saquinavir mesylate, Idinavir, Ritonavir, Amprenavir, Kaletra, and Nelfinavir mesylate;
  • the above-mentioned invasion inhibitor may be Maraviroc, TAK- One or more of 779, T-20, T2635, sifuvirtide, ibbevirin, VIRIP (VIR-576), etc.
  • the above integrated inhibitor may be one or more of Raltegravir, Dolutegravir, and Elvitegravi Kind.
  • the specific therapeutically effective dose level for any particular patient will depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the particular composition employed. The age, weight, general health, sex and diet of the patient; the time of administration, the route of administration and the rate of excretion of the particular active ingredient employed; the duration of treatment; in combination with or in combination with the particular active ingredient employed Drugs; and similar factors well known in the medical field. For example, it is the practice in the art that the dosage of the active ingredient be started from a level lower than that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved.
  • the lipopeptide or polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, the polymer, the composition or the pharmaceutical compound may be administered to a mammal, particularly a human. It is from 0.001 to 1000 mg/kg body weight/day, for example from 0.01 to 100 mg/kg body weight/day, and for example from 0.1 to 10 mg/kg body weight/day.
  • the lipopeptide LP-19 of the present invention has the advantages of broad spectrum, potency and long-lasting effect:
  • LP-19 has strong broad-spectrum activity against HIV-1, HIV-2 and SIV, especially showing strong advantages for HIV-2 and SIV: LP-19 for HIV-1, HIV-2 and SIV Mediated cell membrane fusion has potent inhibitory effects, significantly higher than the three control polypeptides T-20, 2P23 and LP-11 ( Figure 3); LP-19 has both HIV-1 and SIV-mediated cell invasion The potent inhibitory effect was significantly higher than the three control polypeptides T-20, 2P23 and LP-11 ( Figure 4). The inhibitory activity of LP-19 on infectious HIV-1 and HIV-2 was significantly higher than the three control polypeptides T-20, 2P23 and LP-11 (Fig. 5).
  • the inhibitory activity of LP-19 against various subtypes of HIV-1 virus was also significantly higher than the three control polypeptides T-20, 2P23 and LP-11 (Fig. 6).
  • the activity of LP-19 inhibiting T-20 resistant strains was 10613.53 times, 6.18 times and 2.18 times higher than that of T-20, 2P23 and LP-11, respectively; LP-19 inhibited the activity of 2P23 resistant strains compared with T-20 2P23 and LP-11 were 173.63 times higher, 45.09 and 6.83 times higher respectively (Fig. 7).
  • LP-19 showed significant potent and long-acting activity in monkeys.
  • the peak of subcutaneous pathway inhibition was more than 100 times that of T-20, and the peak of intravenous inhibition was more than 2000 times that of T-20.
  • LP-19 inhibited peak serum maximum dilutions of 66 and 40, respectively, even after subcutaneous injection for 60 and 72 hours, similar to T-20 at 2 and 4 hours ( Figure 8).
  • the LP-19 derivatives LP-20 to LP-32 have broad-spectrum activity and are effective against both HIV-1 and HIV-2 and SIV (Fig. 1).
  • Figure 1 shows the structure of a lipopeptide having activity against HIV-1 and/or HIV-2 and/or SIV and its antiviral activity active.
  • Figure 2 shows the structure and function of the HIV fusion protein gp41 and the polypeptide membrane fusion inhibitor.
  • FP refers to a gp41 fusion peptide
  • NHR refers to an N-terminal repeat
  • CHR refers to a C-terminal repeat
  • 6-HB refers to a hexamer helix.
  • the amino acid labeled as black in the polypeptide sequence is the residue forming the M-T hook, and the underlined portion is the pocket binding region (PBD).
  • the amino terminus of all polypeptides or lipopeptides in Figure 2 is linked to an acetyl group as an amino terminal protecting group, and the carboxy terminus of all polypeptides or lipopeptides is linked to an amino group as a carboxy terminal protecting group.
  • Figure 3 shows the inhibition of HIV-1, HIV-2 and SIV mediated cell membrane fusion by LP-19 and control polypeptides.
  • Figure 4 shows the inhibition of entry of HIV-1 and SIV pseudoviral cells by LP-19 and control polypeptides.
  • Figure 5 shows the inhibition of HIV-1 and HIV-2 infection by LP-19 and control polypeptides.
  • Figure 6 shows the inhibition of various subtypes of HIV-1 by LP-19 and a control polypeptide.
  • Figure 7 shows the inhibitory effect of LP-19 and the control polypeptide T-20 on 2P23 resistant strains.
  • Figure 8 shows the antiviral activity of LP-19 and control polypeptide injected into cynomolgus serum.
  • M248, M249, M250, M252, M253 and M254 are the monkey numbers.
  • Figure 9 is a circular dichroism analysis of the 2P23 and LP-19 self-helical structures and their interaction with NHR.
  • NA indicates that the Tm value cannot be accurately determined because the spiral structure is not sufficiently dissociated within the measurement temperature range.
  • E glutamic acid
  • M methionine
  • T threonine
  • W tryptophan
  • K lysine
  • V valine
  • L leucine
  • I is isoleucine
  • C cysteine
  • Q glutamine
  • N is asparagine
  • Y tyrosine
  • S serine
  • T threonine
  • A Alanine
  • Ac is acetyl
  • PEG8 Fmoc-NH-PEG8-CH 2 CH 2 COOH
  • C16 is palmitic acid
  • C18 stearic acid
  • NH 2 is amino
  • Chol is cholesterol
  • Toc is vitamin E.
  • the polypeptide concentration refers to the concentration of the lipopeptide or polypeptide.
  • the lipopeptides of the present invention having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV
  • the most preferred embodiments are the lipopeptides of the above af) and bf).
  • the polypeptide having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV is the P1 (the polypeptide represented by the sequence 1 in the sequence listing, that is, the LP-19, LP-20 in FIG. 1 , a polypeptide represented by amino acid residues 1 to 23 of LP-21, LP-22 and LP-23, which is 2P23), P3 (a polypeptide represented by SEQ ID NO: 2 in the sequence listing, that is, LP in Fig.
  • P5 a polypeptide represented by the sequence 3 in the sequence listing, that is, a polypeptide represented by amino acid residues 1-31 of LP-32 in Fig. 1
  • P7 a polypeptide represented by SEQ ID NO: 4 in the Sequence Listing, that is, a polypeptide represented by amino acid residues 1-34 of LP-29 in Figure 1
  • P9 a polypeptide represented by SEQ ID NO: 5 in the Sequence Listing
  • P11 a polypeptide represented by SEQ ID NO: 6 in the sequence listing, that is, amino acid residues 1 to 25 of LP-24 in Fig.
  • polypeptide represented by a base a polypeptide represented by a base
  • P13 a polypeptide represented by SEQ ID NO: 7 in the sequence listing, that is, a polypeptide represented by amino acid residues 1-31 of LP-28 in Fig. 1
  • P15 a polypeptide represented by amino acid residues 8 in the sequence listing
  • the polypeptide shown that is, the polypeptide represented by amino acid residues 1-30 of LP-30 in Fig. 1
  • P17 sequence 9 in the sequence listing
  • the polypeptide shown that is, the polypeptide represented by amino acid residues 1-27 of LP-26 in Fig. 1 and P19 (the polypeptide shown by SEQ ID NO: 10 in the sequence listing, that is, the first of LP-27 in Fig.
  • the lipophilic compound is palmitic acid (also known as palmitic acid) (C16), stearic acid (C18), cholesterol (cholesterol), dihydrosphingosine or tocopherol.
  • the arm is connected to Fmoc-NH-PEG8-CH 2 CH 2 COOH.
  • the protecting group is an amino terminal protecting group and a carboxy terminal protecting group.
  • the amino terminal protecting group is an acetyl group, and the carboxy terminal protecting group is an amino group.
  • the above-mentioned lipopeptide having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV is designed to be derived from the polypeptide of the HIV fusion protein gp41 CHR region 2P23 and the target sequence of HIV-1, HIV-2 and SIV, ie NHR.
  • the lipophilic compound can bind to the target cell membrane, increasing the concentration of the polypeptide at the site of viral infection, thereby increasing the antiviral activity of the polypeptide.
  • lipophilic compounds also have the effect of binding to plasma proteins such as HSA to increase polypeptide stability and biological half-life.
  • the polypeptide sequence is linked to the lipophilic compound via a flexible linker polyethylene glycol (PEG) to overcome steric hindrance and allow the lipopeptide to function more flexibly ( Figure 2).
  • PEG polyethylene glycol
  • Figure 2 The above lipopeptides having the activity of inhibiting HIV-1 and/or HIV-2 and/or SIV have strong binding ability to NHR target sequences derived from HIV-1, HIV-2 and SIV, and are highly resistant to viruses. Inhibition, exhibits significant long-acting activity in non-human primates (monkeys).
  • This example is designed to have lipopeptides that inhibit HIV-1 and/or HIV-2 and/or SIV activity, and their names are LP-19, LP-20, LP-21, LP-22, LP-23, LP-24, LP-25, LP-26, LP-27, LP-28, LP-29, LP-30, LP-31 and LP-32.
  • the structures of LP-19, LP-20, LP-21, LP-22, LP-23, LP-24 and LP-25 are shown in Figure 1.
  • the design concepts include: (1) The N-terminus of its polypeptide sequence contains MT.
  • the hook structure motif (EMT) enhances the affinity of the polypeptide to the hydrophobic pocket region of NHR; (2) the amino acid whose polypeptide sequence interacts with NHR is derived from the HIV-1/HIV-2/SIV sequence to increase the broad spectrum of the polypeptide.
  • the non-NHR binding amino acid of the polypeptide sequence is replaced by an "i and i+3" diion pair structure (ie, EE--KK structure) to promote the helicity of the polypeptide by forming a "salt bridge” structure; (4) coupling a lipophilic compound to a C-terminus of a polypeptide via a PEG8 linker and a lysine (or cysteine) to confer the ability of the polypeptide to bind to the target cell membrane and/or viral membrane, thereby increasing the virus Invasive local polypeptide concentration; (5) lipophilic compound modified polypeptide has the ability to bind to plasma proteins (such as HSA), thereby improving its biological stability.
  • plasma proteins such as HSA
  • LP-26, LP-27, LP-28, LP-29, LP-30, LP-31 and LP-32 are shown in Figure 1.
  • the polypeptide sequence is identical and the linker arm is absent, and the others are identical to LP-19 or LP-20.
  • the amino terminus of the polypeptides T-20 and 2P23 are both linked to an acetyl group as an amino terminal protecting group, and the carboxy terminus is linked to an amino group as a carboxy terminal protecting group.
  • the structure of these polypeptides is shown in FIG. The synthesis of these polypeptides was carried out by standard solid phase peptide synthesis (Fmoc/tBu strategy) from the carboxy terminus to the amino terminus. All polypeptide sequences are amidated at the C-terminus, acetylated N-terminus according to the conventions of polypeptide synthesis.
  • the condensation method selected for the peptide reaction was DIPC/HOBt method and PyBOP method, and the amino acid and the activating reagent were all used in 3 times equivalent, and the reaction time was 1 hour. The reaction progress was monitored by ninhydrin qualitative color development (Kaiser method). If the condensation reaction of an amino acid is incomplete, the reaction time is appropriately extended or the condensation is repeated once until the desired target peptide is obtained.
  • the polypeptide of interest was cleaved from the resin and the side chain protecting groups were removed (cutting at 30 degrees C for 4 hours).
  • a filtration operation was carried out, and the filtrate was added to a large amount of cold anhydrous diethyl ether to precipitate a polypeptide and centrifuged. After washing several times with diethyl ether, it was dried to obtain a crude polypeptide.
  • the structure was prepared as follows: LP-19, LP-20, LP-21, LP-22, LP-23, LP-24, LP-25, LP-26, LP-27, LP-28 as shown in Fig. 1. , LP-29, LP-30, LP-31 and LP-32.
  • Palmitic acid (LP-19, LP-26 to LP-30), stearic acid (LP-20, LP-24, LP-25, LP-31, LP-32), dihydro (neuro) sphingosine ( The modification of the polypeptide by LP-22) or vitamin E (LP-23) is accomplished by amidation of the side chain amino group of the C-terminal Lys of the polypeptide (refer to background art in Reference 15: Chong H, Wu X, Su Y, He Y. Development of potent and long-acting HIV-1 fusion inhibitors. AIDS 2016, 30(8): 1187-1196).
  • Chemical reagents used such as Rink Amide MBHA resin, Fmoc-PEG4-OH (Fmoc-15-amino-4, 7, 10, 13-tetraoxapentadecacanoic acid), various Fmoc amino acids, palmitoyl chloride, N, N '-Diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), trifluoroacetic acid (TFA), ethanedithiol (EDT), ninhydrin, hexahydropyridine (PIPE) , phenol, N, N'-dimethylformamide (DMF), chromatographically pure acetonitrile, etc. are all purchased from major chemical reagent suppliers. No further purification before use.
  • Rink Amide MBHA resin (substitution constant 0.34 mmol/g) was used as a starting material, and it was synthesized from the C terminal to the N terminal.
  • the Fmoc protecting group on the Rink resin was removed with 25% hexahydropyridine/DMF (volume ratio), and then grafted with a resin with 2 times equivalent of Fmoc-Lys(Dde)-OH/HOBt/DIC to introduce the first amino acid at the C-terminus. Residues. Thereafter, the N-terminal Fmoc protecting group was again removed with 25% hexahydropyridine/DMF (volume ratio) to make the N-terminus a free amino group. The two PEG4 and each amino acid residue are sequentially linked in this order.
  • the starting materials and amounts used correspond to Fmoc-PEG4-OH (1.5 eq), Fmoc-PEG4-OH (1.5 eq), Fmoc-Lys(Boc)-OH (3 eq), Fmoc-Leu-OH (3 eq), Fmoc- Leu-OH (3 eq), Fmoc-Glu(OtBu)-OH (3 eq), Fmoc-Glu(OtBu)-OH (3 eq), Fmoc-Ile-OH (3 eq), Fmoc-Lys(Boc)-OH (3 eq ), Fmoc-Lys(Boc)-OH (3 eq), Fmoc-Glu(OtBu)-OH (3 eq), Fmoc-Leu-OH (3 eq), Fmoc-Glu(OtBu)-OH (3 eq), Fmoc-Glu (OtBu)-OH (3 eq), Fmoc-Leu
  • N-terminal acetylation end-capping (3 times equivalent of Ac 2 O, 6-fold equivalent of diisopropylethylamine) was carried out to complete the synthesis of the main chain.
  • the reaction time of each step was as follows: deprotection for 8 minutes, twice; grafting ordinary amino acid for 60 minutes, grafting PEG4 for 180 minutes.
  • the resin was treated with 2% hydrazine hydrate/DMF solution (volume ratio) to remove the protective group of the side chain of the C-terminal Lys, and then mixed with 3 times equivalent of palmitoyl chloride and 6 times equivalent of diisopropylethylamine with C-terminal Lys.
  • the side chain amino group is subjected to an amidation reaction (60 minutes) to effect palmitoylation modification on the C-terminal Lys residue.
  • the resin After each step of the above reaction, the resin needs to be washed with DMF six times or more, and the progress of the reaction is monitored by ninhydrin qualitative color development (Kaiser method). If the condensation reaction of an amino acid is incomplete, the condensation is repeated once until the desired one is obtained. Target peptide.
  • Purification and characterization of peptides and lipopeptides Purification of crude peptides and crude lipopeptides was performed on a reversed-phase high performance liquid chromatograph. The column used was a reverse phase C18 silica gel of 10 micron particle size with a pore size of 100 angstroms and a column size of 50 x 250 mm.
  • Chromatographic operating conditions linear gradient elution, the eluent consists of mobile phase A and mobile phase B.
  • the mobile phase A was an aqueous solution of trifluoroacetic acid and acetonitrile, the volume percent concentration of trifluoroacetic acid was 0.05%, and the volume percent concentration of acetonitrile was 2%.
  • Mobile phase B was a 90% (by volume percent) aqueous solution of acetonitrile.
  • the chemical structure was characterized by MALDI-TOF mass spectrometry, and the purity was given by analytical high performance liquid chromatography (flow rate: 1 ml per minute).
  • the model of analytical high performance liquid chromatography Shimadzu CBM-10A VP PULS, the type of column used: Agela 4.6*250mm C18.
  • Chromatographic operating conditions linear gradient elution, the eluent consists of mobile phase A and mobile phase B.
  • the mobile phase A was an aqueous solution of trifluoroacetic acid and acetonitrile, the volume percent concentration of trifluoroacetic acid was 0.05%, and the volume percent concentration of acetonitrile was 2%.
  • the mobile phase B was an aqueous solution of trifluoroacetic acid and acetonitrile, the volume percent concentration of trifluoroacetic acid was 0.05%, and the volume percent concentration of acetonitrile was 90%.
  • the linear gradient elutes from 25% B to 45% B for 20 minutes.
  • the obtained polypeptide and lipopeptide pure product were characterized by analytical reversed-phase high performance liquid chromatography to indicate that LP-19, LP-20, LP-21, LP-22, LP-23, LP-24, LP-25, LP- 26.
  • the purity of LP-27, LP-28, LP-29, LP-30, LP-31LP-32, T-20 and 2P23 are all greater than 95%.
  • HEK293T cells (abbreviated as 239T cells) were purchased from the American Model Culture Collection (ATCC); U87CD4+CXCR4+ cells were provided by the US NIH AIDS Reagents and References Project (Cat. No.
  • HIV-2 strain ROD molecules The cloned plasmid pROD was kindly provided by Professor Nuno Taveira of the University of Lisbon, Portugal; the plasmids expressing the SIV strains SIV pbj and SIV 239 envelope proteins (pSIVpbj-Env and pSIV239, respectively) were donated by Professor Xu Jianqing from Fudan University; the fluorescent reporter system plasmid DSP 1- 7 and DSP 8-11 were donated by Professor Zene Matsuda of the University of Tokyo, Japan; the plasmid expressing the envelope protein of HIV-1 strain NL4-3 is inserted into the vector of the envelope protein (ENV) encoding gene of HIV-1 strain NL4-3.
  • ENV envelope protein
  • 239T cells effector cells
  • U87CD4+CXCR4+ target cells
  • the two were cultured overnight at 37 ° C and 5% CO 2 .
  • the plasmid expressing the envelope protein (Env) and the DSP 1-7 plasmid were mixed 1:1 and transfected into 239T cells, and the DSP 8-11 plasmid was transfected into U87CD4+CXCR4+ cells.
  • the DSP 8-11 plasmid-transfected U87CD4 + CXCR4+ cells were resuspended in 300 ⁇ l of pre-warmed culture medium and 0.05 ⁇ l of EnduRen live cell bottom was added. (Promega). After the test substance T-20, 2P23, LP-11 or LP-19 was dissolved in dimethyl sulfoxide (DMSO) and diluted with the cell culture medium, the effector cells added to the 96-well culture plate were diluted by a factor of three. Then, transfer 75 ⁇ l of target cells to the effector cells.
  • DMSO dimethyl sulfoxide
  • the HIV recombinant pseudovirus system was used to evaluate the inhibitory effect of the polypeptide on the entry of the virus into the target cell.
  • the basic steps include (1) preparation of a pseudovirus: a plasmid expressing the envelope protein of HIV-1 NL4-3, SIV pbj or SIV 239 strain and the HIV backbone plasmid pSG3 ⁇ ENV (expressing all proteins except the ENV in the HIV genome) It was provided by the NIH AIDS Reagents and References Project of the United States, catalog number 11051), transfected into 293T cells at a mass ratio of 1:2, and a control that only transfected the same amount of pSG3 ⁇ ENV. After incubating for 6 hours at 37 ° C in a 5% CO 2 cell incubator, the solution was changed, and then incubation was continued for 48 hours to secrete the pseudovirus into the supernatant.
  • a pseudovirus a plasmid expressing the envelope protein of HIV-1 NL4-3, SIV pbj or SIV 239 strain and the HIV backbone plasmid pSG3 ⁇ ENV (expressing all proteins except the ENV in the HIV genome
  • TZM-bl cells were trypsinized and counted, and the cells were diluted to 1 ⁇ 10 5 /ml with DMEM complete medium, and 100 ⁇ l of cells (containing 15 ⁇ g/ml DEAE-dextran) per well were added at 37 ° C. 5% CO 2 for 48 hours.
  • the 96-well plate was then removed from the cell culture incubator, the supernatant was aspirated from the well, and 30 ⁇ l of the cell lysate was added, and after 10 minutes, 100 ⁇ l of luciferase assay reagent was added. Pipette 100 ⁇ l of liquid from each well and add it to the corresponding 96-well white plate to read the luminescence value on a microplate luminometer.
  • the virus titer was calculated using the Reed-Muench method.
  • (3) Antiviral activity test The test substance (T-20, 2P23, LP-11 or LP-19 (dissolved in DMSO and diluted with cell culture solution) was diluted into a 96-well plate by a ratio of (3 times) dilution. The final volume was 50 microliters, in which 50 microliters of DMEM medium was used instead of the test substance as a negative control.
  • the cells were incubated for 6 hours at 37 ° C in a 5% CO 2 cell incubator and then changed for 48 hours. .
  • the virus titration is carried out in the same manner as the above-mentioned HIV pseudovirus.
  • the test substance T-20, 2P23, LP-11 or LP-19 (dissolved in DMSO and diluted with cell culture medium) was diluted in a ratio of (3 times) into a 96-well plate.
  • the final volume was 50 microliters with 50 microliters of DMEM medium instead of the test substance as a negative control.
  • the relative fluorescence units (RLU) of each well were determined using a luciferase assay reagent (Promega). The % inhibition rate and the IC 50 value were calculated.
  • LP-19 had potent inhibitory effects on HIV-1, HIV-2 and SIV-mediated cell membrane fusion, which was significantly higher than the three control polypeptides.
  • the inhibitory IC 50 of LP-19 against HIV-1 strain NL4-3 mediated cell fusion was 0.14 nM, while the inhibitory IC 50 values of T-20, 2P23 and LP-11 were 7.17 nM, 0.28 nM and 0.78 nM; LP-19 inhibited ROD-mediated cell fusion of HIV-2 strain with IC 50 of 2.27 nM, while T-20, 2P23 and LP-11 inhibited IC 50 values of 569.8 nM and 12.25 nM, respectively.
  • SIV pbj cell fusion mediated inhibition of LP-19 IC 50 of 0.67nM, and the IC 50 values of inhibition of T-20,2P23 thereof and LP-11 were 5.44nM, 1.91nM, and 4.18nM ;
  • LP-19 SIV 293 cell fusion mediated inhibition IC 50 of 2.28nM, and the IC 50 values of inhibition of T-20,2P23 thereof and LP-11 were 219nM, 2.64nM and 11.91nM.
  • LP-19 has potent inhibitory effects on HIV-1 and SIV-mediated cell invasion, significantly higher than the three control polypeptides T-20, 2P23 and LP-11.
  • AIDS is caused mainly by HIV-1 worldwide, and a variety of subtypes, including AD, FH, J and K subtypes, are produced due to viral mutations. Among them, subtypes A, B and C are the main viruses causing the world AIDS epidemic. In China, the prevalence of B/C and A/E recombinant viruses is dominant.
  • a group of 29 HIV-1 pseudoviruses including international representative strains and HIV strains currently prevalent in China, were prepared, including 3 subtypes A and 6 subtype B.
  • T-20 is currently the only HIV membrane fusion inhibitor approved for clinical treatment. However, its activity is not only significantly lower than that of a new generation of peptides, but also easily induces drug-resistant mutations, leading to the failure of clinical antiviral therapy.
  • 2P23 is a newly designed short-chain peptide containing MT hook structure with only 23 amino acids. It mainly targets the NHR hydrophobic pocket structure of fusion protein gp41, and has good inhibitory activity against HIV-1, HIV-2 and SIV. However, some resistance to mutations induced by some short peptides also showed some cross-resistance.
  • the inventors prepared pseudoviruses carrying NHR mutations containing T-20 and 2P23 corresponding resistance sites (Fig. 7). These plasmids for the preparation of pseudoviruses were obtained by site-directed mutagenesis by the inventors based on the plasmid pNL4-3-Env expressing the envelope protein of HIV-1 strain NL4-3, and were preserved and routinely performed by Professor He Yuxian of the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. use.
  • the T-20 resistant strains in Figure 7 are (Chong H, Yao X, Zhang C, Cai L, Cui S, Wang Y, He Y.
  • the HIV-1 NL4-3 mutant strain in Table 3 the subscript of the name of the strain in Figure 7 of the present invention is the table in the literature.
  • the 2P23 resistant strains were (Su Y, Chong H, Xiong S, Qiao Y, Qiu Z, He Y. Genetic pathway of HIV-1 resistance to novel fusion inhibitors targeting the Gp41pocket. J Virol.
  • the activity of LP-19 inhibiting T-20 resistant strains was 10613.53 times, 6.18 times and 2.18 times higher than that of T-20, 2P23 and LP-11, respectively.
  • the average IC 50 of LP-19 inhibiting 2P23 resistant strains were respectively It is 134.4 nM, 36.52 nM, 5.53 nM and 0.81 nM. It can be seen that the activity of LP-19 inhibiting 2P23 resistant strains was 165.93 times, 45.09 and 6.83 times higher than that of T-20, 2P23 and LP-11, respectively.
  • LP-19 is a broad spectrum of highly active antiviral lipopeptides.
  • LP-19 and the control peptides T-20 and 2P23 were injected into monkeys by subcutaneous or intravenous injection, and blood samples were taken at different time points to determine serum in vitro.
  • Antiviral activity The method can not only understand the in vivo antiviral activity of the test substance, but also help to analyze the stability and half life of the test substance in vivo.
  • the specific method is as follows: 6 experimental macaques (rhesus monkeys) were selected, half male and half female, aged 3-4 years old, weighing 3.4-4.7 kg.
  • test substance T-20, 2P23 or LP-19 (all dissolved in sterile distilled water) at a dose of 3 mg/kg (kg/kg)
  • 0.4 ml of venous blood samples were taken at 1, 2, 4, 6, 8, 12, 18, 24, 36, 48, 60, and 72 hours after the injection, and the serum was separated according to a conventional method.
  • LP-19 was additionally injected intravenously with LP-19 (dissolved in sterile distilled water) at a dose of 3 mg/kg body weight. The interval between each experiment was more than 2 weeks to ensure that there was no residue of the last injection of the test substance.
  • the activity of the serum-inhibiting HIV-1 pseudovirus NL4-3 mutant D36G was examined by an antiviral assay according to the method of Example 3.
  • the serum was diluted 3 times.
  • the results of the experiment are shown in Fig. 8.
  • Subcutaneous injection of T-20 showed a peak of inhibition at 2 and 4 hours, and the maximum dilutions of serum inhibiting 50% of NL4-3 infection were 45-fold and 46-fold, respectively; subcutaneous injection of short peptide 2P23 was The peaks of inhibition were 1 and 2 hours, and the maximum dilution ratio of serum was 60 times and 68 times, respectively.
  • the subcutaneous injection of short peptide LP-19 showed peak inhibition at 6 and 8 hours, and the maximum dilution ratio of serum was 5396 times and 4720 times, respectively.
  • Intravenous injection of LP-19 showed a peak of inhibition at 1 and 2 hours, and the maximum dilution factor of serum was 99107 times and 76346 times, respectively. It can be seen that LP-19 is markedly potent and long-acting in monkeys.
  • the peak of subcutaneous pathway inhibition is more than 100 times that of T-20, and the peak of intravenous inhibition is more than 2000 times that of T-20.
  • LP-19 inhibited peak serum maximum dilutions of 66 and 40, respectively, even after subcutaneous injection for 60 and 72 hours, similar to the performance of T-20 at 2 and 4 hours.
  • the secondary structure and helical stability (Tm value) of the peptide were determined by circular dichroism (CD).
  • the experimental method was referenced [14].
  • the circular dichroic instrument is Nissan Jasco-815.
  • the self-helical content and Tm value of LP-19 and its template 2P23 at different concentrations (20, 40, 80, 160 and 320 ⁇ M, respectively) were determined.
  • the helix content and Tm value of 6-HB formed by LP-19 or 2P23 and NHR polypeptide N36 (Ac-SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH2) were then determined.
  • 2P23, LP-19 and N36 were each dissolved in phosphate buffer (PBS, pH 7.2), and the concentration was determined according to ultraviolet absorption at 280 nm.
  • concentration was determined according to ultraviolet absorption at 280 nm.
  • the interaction between the polypeptides and the helix content were determined based on the CD signal.
  • the stability of the six-helix structure formed by the polypeptide inhibitor and N36 was determined by CD temperature scanning.
  • the unmodified polypeptide 2P23 as a control contained a typical ⁇ -helical structure (A in Fig. 9), and the spiral contents at the concentrations of 20 ⁇ M, 40 ⁇ M, 80 ⁇ M, 160 ⁇ M, and 320 ⁇ M were 76.4%, 81.4%, and 83.8%, respectively. 84% and 79.7%; their Tm values at concentrations of 20 ⁇ M, 40 ⁇ M, 80 ⁇ M, and 160 ⁇ M were 48.1 degrees Celsius, 54.7 degrees Celsius, 59.9 degrees Celsius, and 67.8 degrees Celsius, respectively (B in Figure 9).
  • the helix content of lipopeptide LP-19 at concentrations of 20 ⁇ M, 40 ⁇ M, 80 ⁇ M, 160 ⁇ M and 320 ⁇ M was 70%, 71.1%, 71.9%, 76.2% and 71%, respectively (C in Figure 9); however, at the highest temperature determined (98 degrees Celsius), the helix of LP-19 could not be fully unscrewed under various concentrations of CD, indicating that palmitic acid modification can significantly increase the helical stability of the polypeptide (D in Figure 9).
  • LP-19 lipopeptide has the advantages of broad spectrum, potency and long-lasting effect.
  • a new group of lipopeptides was synthesized by substituting other lipophilic compounds for palmitic acid in LP-19 (Fig. 1).
  • LP-20, LP-24 and LP-25 are modified with stearic acid (C18)
  • LP-21 is modified with cholesterol (Chol)
  • LP-22 is modified with dihydro (neuro) sphingosine (Dih)
  • LP -23 is modified with vitamin E (Toc).
  • lipopeptides LP-26 to LP-32) containing no PEG8 but substituted with amino acid sequences of different lengths were also synthesized.
  • the lipopeptide, derivative thereof, or pharmaceutically acceptable salt thereof provided by the present invention, the polymer, the composition or the pharmaceutical compound can be used for HIV (HIV-1 and/or HIV-2) And/or treatment and/or prevention of SIV infection.
  • the lipopeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, the multimer, the composition or the pharmaceutical compound can be directly administered to a patient as a medicament, or with a suitable The vehicle or vehicle is mixed and administered to the patient for the purpose of treatment and/or HIV infection.

Abstract

提供了抑制HIV的脂肽，该脂肽包括具有抗病毒活性的多肽，与多肽的羧基末端相连的连接臂，以及与连接臂相连的亲脂性化合物；或者该脂肽包括具有抗病毒活性的多肽，以及与多肽的羧基末端相连的亲脂性化合物。还提供了该脂肽的衍生物、药物组合物及其用途。

Description

广谱抑制HIV的脂肽、其衍生物、其药物组合物及其用途 技术领域
本发明涉及生物医药领域中广谱抑制HIV的脂肽、其衍生物、其药物组合物及其用途。
背景技术
引起艾滋病(AIDS)的人免疫缺陷病毒(HIV)分为1型和2型两种。目前全球HIV感染人数大约3600万，HIV-1是主要的病原体，约有100-200万人由HIV-2感染引起，大多发生在西非地区。然而，HIV-2已逐渐传播到欧洲、亚洲和北美等地区，对人类健康造成严重威胁[1]。疫苗是预防艾滋病的最好的手段，但有效的艾滋病疫苗近期恐难有重大突破。因此，研发阻断病毒不同复制阶段的药物是目前防控艾滋病的重点策略。由于目前临床应用的艾滋病治疗药物都是根据HIV-1设计，导致许多药物对HIV-2的活性有限，无法用于HIV-2感染的临床治疗。
HIV进入靶细胞由其表面的包膜糖蛋白(Env)介导[2]。该蛋白由表面亚基gp120和跨膜亚基gp41通过非共价键连接而成，在天然状态下呈三聚体结构。gp120的主要功能是与细胞受体CD4和辅助受体(如趋化因子受体CCR5或CXCR4等)结合，而gp41主要介导病毒和细胞的膜融合。研究发现，gp41膜外区包含几个重要的功能区，包括疏水性融合肽(FP)、N-末端螺旋重复序列(NHR)、C-末端螺旋重复序列(CHR)。早在1997年，通过解析来源于NHR和CHR的多肽复合物的晶体结构，发现gp41的核心结构为六股α-螺旋束(six helix bundle,6-HB)，其中三个NHR组成的N-螺旋通过在a和d位置的氨基酸残基相互作用形成位于中心的螺旋三聚体，其e和g位置的氨基酸残基则暴露于中心螺旋体的外围，并与三个CHR组成的C-螺旋的a和d位置相互作用[3,4]。C-螺旋以反向平行的方式分别结合在三个N-螺旋形成的沟槽中。基于gp41的三维结构信息提出了HIV-1膜融合机制：当gp120与靶细胞上的受体结合后发生显著的构象变化，从而使gp41的融合肽暴露出来并插入靶细胞膜内，CHR与NHR发生反向结合，形成稳定的6-HB结构，将病毒膜与靶细胞膜拉近而发生融合，从而介导HIV进入靶细胞内。晶体结构揭示，NHR的C端部分形成明显的疏水深口袋结构(pocket),CHR的N端疏水口袋结合区(PBD)插入NHR疏水口袋，对稳定6-HB结构以及病毒的感染活性至关重要，被认为是抗HIV药物的新靶点[5,6]。
来源于gp41NHR和CHR的多肽具有显著的抗HIV活性，主要是通过与对应的NHR或CHR结合而竞争性地阻断病毒本身6-HB的形成，从而阻断病毒-细胞膜融合过程[7,8]。于2003年获得美国FDA批准的艾滋病治疗药物T-20(Enfuvirtide，Fuzeon)即是来源于CHR的含有36个氨基酸残基的多肽。T-20是第一个也是目前唯一批准用于临床治疗的病毒膜融合抑制剂，但其容易诱导耐药产生，而且对HIV-2 活性很差，极大地限制了它的广泛应用[9]。长度为39个氨基酸的多肽T1249是T20的第二代产品，具有较高的抑制HIV-1和HIV-2的能力，但由于其剂型和成本问题而停止了临床试验。所以，许多研究致力于研发新一代HIV膜融合抑制多肽。由于T-20不含重要的NHR口袋结合序列(PBD)，另一CHR多肽C34被广泛作为新型抑制剂的设计模板[7]。然而，多数新设计的多肽同T-20和T1249一样，都具有较长的氨基酸序列，如T2635含有38个氨基酸，SC35EK含有35个氨基酸。中国临床试验中的西夫韦肽(SFT)和艾博韦肽(ABT)分别有36和34个氨基酸。一些利用HIV-2和/或SIV序列设计的多肽也同样较长，如P3和C34EHO均含有34个氨基酸[10]。同时，多肽类药物的一个明显缺陷是其较短的生物半衰期，如T-20的半衰期仅为3.8小时，因此临床上病人治疗需要采用大剂量的频繁注射(90mg/次，2次/天)。近年来Chong H等聚焦于HIV融合蛋白gp41的结构与功能及抑制剂研究，提出了基于“M-T钩子结构”和“盐桥螺旋结构”设计靶向NHR疏水口袋短小多肽HIV膜融合抑制剂的新概念[11,12]。最近，Xiong S等通过导入HIV-2/SIV序列所设计的多肽2P23仅有23个氨基酸，不但抗病毒活性高，而且比较广谱，对HIV-1、HIV-2以及猴免疫缺陷病毒(SIV)都具有很强的抑制活性[13]。近年的研究也表明，通过对多肽进行脂类化学修饰，即所谓的“脂肽”(lipopeptide),不但能提高多肽的抗病毒靶向性和活性，也能显著改善多肽的稳定性和生物半衰期，例如胆固醇修饰的C34(C34-Chol)[14]及棕榈酸(C16)修饰的HP23短肽(LP-11)[15]。这些研究进展都为设计新的HIV膜融合抑制剂奠定了坚实的理论基础和技术路线。
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技术问题
本发明所要解决的一个技术问题是如何提高具有抑制HIV-1和/或HIV-2和/或SIV活性的多肽对HIV-1和/或HIV-2和/或SIV抗病毒活性，和/或延长其抗病毒活性持续时间。
技术解决方案
为了解决以上技术问题，本发明提供了具有抗HIV-1和/或HIV-2和/或SIV活性的脂肽。
本发明所提供的具有抗HIV-1和/或HIV-2和/或SIV活性的脂肽(化合物)为下述a)或b)：
a)所述脂肽包括具有抗病毒活性的多肽，与所述多肽的羧基末端相连的连接臂，与所述连接臂相连的氨基酸X残基和与所述氨基酸X残基相连的亲脂性化合物；所述氨基酸X为K、C、S、T或Y；
b)所述脂肽包括具有抗病毒活性的多肽，与所述多肽的羧基末端相连的亲脂性化合物；
所述a)或b)中，所述病毒为下述v1-v7中的任一：
v1、HIV-1、HIV-2和SIV；
v2、HIV-1和HIV-2；
v3、HIV-1和SIV；
v4、HIV-2和SIV；
v5、HIV-1；
v6、HIV-2；
v7、SIV。
上述具有抗HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述抗病毒活性也可称为抑制病毒活性，具体可为抑制病毒进行细胞融合和/或抑制病毒侵入细胞和/或抑制病毒复制。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述a)或b)中，所述连接臂可为柔性连接臂，如为Fmoc-NH-PEGn-CH2CH2COOH，n为2、3、4、6、8、10、11或12，所述连接臂也可被本领域公知的其他柔性连接臂所替代；和/或，
所述亲脂性化合物可为含8到20个碳原子的脂肪酸(fatty acid)或胆固醇(cholesterol)、二氢(神经)鞘氨醇(dihydrosphingosine)；所述亲脂性化合物可为维生素E(tocopherol)等。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述含8到20个碳原子的脂肪酸可为棕榈酸(又名软脂酸)(C16)或硬脂酸(C18)。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述具有抑制HIV-1和/或HIV-2和/或SIV活性的多肽中的所有氨基酸可为L型氨基酸，其中的一个或多个氨基酸也可以用构象为D-型的氨基酸、人工修饰的氨基酸、自然界存在的稀有氨基酸等进行替换，以提高多肽的生物利用度、稳定性和/或抗病毒活性。其中D-型氨基酸是指与组成蛋白质的L型氨基酸相对应的氨基酸；人工修饰的氨基酸指经过甲基化、磷酸化等修饰的组成蛋白质的常见L型氨基酸；自然界存在的稀有氨基酸包括组成蛋白质的不常见氨基酸和不组成蛋白质的氨基酸，例如5-羟基赖氨酸、甲基组氨酸、γ氨基丁酸、高丝氨酸等。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述a)或b)中，所述多肽为P1-P20中的任一种：
P1、序列表中序列1所示的多肽；即图1中的LP-19、LP-20、LP-21、LP-22和LP-23的第1-23位氨基酸残基所示的多肽；
P2、在序列表中序列1所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P3、序列表中序列2所示的多肽；即图1中的LP-31的第1-31位氨基酸残基所示的多肽；
P4、在序列表中序列2所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P5、序列表中序列3所示的多肽；即图1中的LP-32的第1-31位氨基酸残 基所示的多肽；
P6、在序列表中序列3所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P7、序列表中序列4所示的多肽；即图1中的LP-29的第1-34位氨基酸残基所示的多肽；
P8、在序列表中序列4所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P9、序列表中序列5所示的多肽；即图1中的LP-25的第1-25位氨基酸残基所示的多肽；
P10、在序列表中序列5所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P11、序列表中序列6所示的多肽；即图1中的LP-24的第1-25位氨基酸残基所示的多肽；
P12、在序列表中序列6所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P13、序列表中序列7所示的多肽；即图1中的LP-28的第1-31位氨基酸残基所示的多肽；
P14、在序列表中序列7所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P15、序列表中序列8所示的多肽；即图1中的LP-30的第1-30位氨基酸残基所示的多肽；
P16、在序列表中序列8所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P17、序列表中序列9所示的多肽；即图1中的LP-26的第1-27位氨基酸残基所示的多肽；
P18、在序列表中序列9所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
P19、序列表中序列10所示的多肽；即图1中的LP-27的第1-28位氨基酸残基所示的多肽；
P20、在序列表中序列10所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述衍生多肽由20到34个氨基酸残基组成。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述a)或b)中，为了提高稳定性，所述脂肽还包括氨基端保护基和/或羧基端保护基，所述氨基端保护基连接在所述多肽的氨基末端上，所述羧基端保护基连接在所述多肽的羧基末端上。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述脂肽为af)或bf)：
af)、由所述具有抗病毒活性的多肽，与所述多肽的羧基末端相连的连接臂，与所述连接臂相连的氨基酸X残基和与所述氨基酸X残基相连的亲脂性化合物和保护基组成；所述氨基酸X为K、C、S、T或Y；所述保护基为氨基端保护基和/或羧基端保护基，所述氨基端保护基连接在所述脂肽的氨基末端上，所述羧基端保护基连接在所述脂肽的羧基末端；
bf)、由所述具有抗病毒活性的多肽，与所述多肽的羧基末端相连的亲脂性化合物和保护基组成；所述保护基为氨基端保护基和/或羧基端保护基，所述氨基端保护基连接在所述脂肽的氨基末端上，所述羧基端保护基连接在所述脂肽的所述羧基末端。
本发明的氨基端保护基可为乙酰基、氨基、马来酰基、琥珀酰基、叔丁氧羰基或苄氧或其他疏水基团或大分子载体基团中的任一基团；所述羧基端保护基可为氨基、羧基、酰胺基或叔丁氧羰基或其他疏水基团或大分子载体基团中的任一基团。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，所述连接臂为Fmoc-NH-PEG8-CH2CH2COOH。
Fmoc-NH-PEG8-CH2CH2COOH的英文名称为1-(9H-fluoren-9-ylmethyl)ester或5,8,11,14,17,20,23,26-Octaoxa-2-azanonacosanedioic acid，C34H49NO12)。在具体脂肽化学合成过程中，Fmoc-NH-PEG8-CH2CH2COOH可由两个Fmoc-NH-PEG4-CH2CH2COOH(英文名称为Fmoc-15-amino-4,7,10,13-tetraoxapentadecacanoic acid)串联而成。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽具体可为图1中的LP-19至LP-32这14个脂肽中的任一个脂肽。
上述P1-P20中的任一种的多肽、其药用盐、或其衍生物也属于本发明的保护范围。
所述多肽的衍生物具体可为下述1)-5)中的至少一种：
1)所述多肽的氨基端连接氨基端保护基和/或所述多肽的羧基端连接羧基端保护基得到的连接物；
2)所述多肽的羧基端连接寡肽或亲脂性化合物得到的连接物；
3)所述多肽的氨基端连接寡肽或亲脂性化合物得到的连接物；
4)所述多肽的氨基端和羧基端均连接寡肽或亲脂性化合物得到的连接物；
5)所述多肽被蛋白质、聚乙二醇、马来酰亚胺修饰得到的修饰物。
PM1或PM2的多聚体也属于本发明的保护范围：
PM1、由所述脂肽、其药用盐、或其衍生物形成的多聚体；
PM2、由所述多肽、其药用盐、或其衍生物形成的多聚体。
下述组合物也属于本发明的保护范围：一种组合物，其包含C1)和C2)： C1)为C11)、C12)或/和C13)；所述C11)为所述脂肽、其衍生物、或其可药用盐；所述C12)为所述多肽、其衍生物、或其可药用盐；所述C13)为所述多聚体；
C2)药学上可接受的载体或辅料；
所述组合物具有下述F1)-F5)中的至少一种功能：
F1)抗病毒；
F2)治疗和/或预防和/或辅助治疗病毒感染所致疾病(如艾滋病)；
F3)抑制病毒进行细胞融合；
F4)抑制病毒侵入细胞；
F5)抑制病毒复制；
F1)-F5)中,所述病毒为下述v1-v7中的任一：
v1、HIV-1、HIV-2和SIV；
v2、HIV-1和HIV-2；
v3、HIV-1和SIV；
v4、HIV-2和SIV；
v5、HIV-1；
v6、HIV-2；
v7、SIV。
上述C11)、C12)、C13)或/和C14)在制备E1)-E5)中至少一种产品中的应用也属于本发明的保护范围：
所述C14)为所述组合物；
所述E1)为抗病毒的产品，如药物或疫苗；
所述E2)为治疗和/或预防和/或辅助治疗病毒感染所致疾病(如艾滋病)的产品，如药物或疫苗；
所述E3)为抑制病毒进行细胞融合的产品，如药物或疫苗；
所述E4)为抑制病毒侵入细胞的产品，如药物或疫苗；
所述E5)为抑制病毒复制的产品，如药物或疫苗；
所述E1)-E5)中，所述病毒为下述v1-v7中的任一：
v1、HIV-1、HIV-2和SIV；
v2、HIV-1和HIV-2；
v3、HIV-1和SIV；
v4、HIV-2和SIV；
v5、HIV-1；
v6、HIV-2；
v7、SIV。
为了解决以上技术问题，本发明提供了药用化合物。
本发明所提供的药用化合物为所述C11)、所述C12)或所述C13)。
上述药用化合物中，所述药用化合物具有下述F1)-F5)中的至少一种功能：
F1)抗病毒；
F2)治疗和/或预防和/或辅助治疗病毒感染所致疾病；
F3)抑制病毒进行细胞融合；
F4)抑制病毒侵入细胞；
F5)抑制病毒复制；
F1)-F5)中,所述病毒为下述v1-v7中的任一：
v1、HIV-1、HIV-2和SIV；
v2、HIV-1和HIV-2；
v3、HIV-1和SIV；
v4、HIV-2和SIV；
v5、HIV-1；
v6、HIV-2；
v7、SIV。
如下治疗或/和预防病毒感染动物的方法也属于本发明的保护范围：
治疗或/和预防病毒感染动物的方法，包括给受体动物施用所述C11)、所述C12)、所述C13)或/和C14)以抑制病毒感染动物；
所述C14)为所述组合物；
所述病毒为下述v1-v7中的任一：
v1、HIV-1、HIV-2和SIV；
v2、HIV-1和HIV-2；
v3、HIV-1和SIV；
v4、HIV-2和SIV；
v5、HIV-1；
v6、HIV-2；
v7、SIV。
本发明所提供的脂肽或多肽、其衍生物、或其可药用盐，所述多聚体，所述组合物或所述药用化合物，可以用于HIV(HIV-1和/或HIV-2)和/或SIV感染的治疗和/或预防。在实际应用中，可以将本发明的脂肽或多肽、其衍生物、或其可药用盐，所述多聚体，所述组合物或所述药用化合物作为药物直接给予病人、或者与适宜的载体或赋形剂混合后给予病人，以达到治疗和/或预防HIV感染的目的。这里的载体材料包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。其中优选的是水溶性载体材料。使用这些材料可以制成多种剂型，包括但不限于片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。 可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将单位给药剂型制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。为了将单位给药剂型制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将单位给药剂型制成注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。使用上述剂型可以经注射给药，包括皮下注射、静脉注射、肌肉注射和腔内注射等；腔道给药，如经直肠和阴道；呼吸道给药，如经鼻腔；粘膜给药。上述给药途径优选的是注射给药。
本发明的脂肽或多肽、其衍生物、或其可药用盐，所述多聚体，所述组合物或所述药用化合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重及个体反应，所用的具体活性成分，给药途径及给药次数等。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药。
本发明的脂肽或多肽、其衍生物、或其可药用盐，所述多聚体，所述组合物或所述药用化合物可以直接单独用于HIV感染者的治疗和预防，也可以与一种或多种抗HIV药物联合使用，以达到提高整体治疗效果的目的。这些抗HIV药物包括但不限于逆转录酶抑制剂、蛋白酶抑制剂、侵入抑制剂、整合抑制剂和成熟抑制剂等。上述的逆转录酶抑制剂可以是AZT、3TC、ddI、d4T、ddT、TDF、Abacavir、Nevirapine、 Efavirenz和Delavirdine等的一种或几种；上述的蛋白酶抑制剂可以是Saquinavir mesylate、Idinavir、Ritonavir、Amprenavir、Kaletra和Nelfinavir mesylate等的一种或几种；上述的侵入抑制剂可以是Maraviroc、TAK-779、T-20、T2635、西夫韦肽、艾博韦肽、VIRIP(VIR-576)等的一种或几种；上述的整合抑制剂可以是Raltegravir、Dolutegravir和Elvitegravi等的一种或几种。
对于任何具体的患者，具体的治疗有效剂量水平须根据多种因素而定，所述因素包括所治疗的障碍和该障碍的严重程度；所采用的具体活性成分的活性；所采用的具体组合物；患者的年龄、体重、一般健康状况、性别和饮食；所采用的具体活性成分的给药时间、给药途径和排泄率；治疗持续时间；与所采用的具体活性成分组合使用或同时使用的药物；及医疗领域公知的类似因素。例如，本领域的做法是，活性成分的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。一般说来，本发明的脂肽或多肽、其衍生物、或其可药用盐，所述多聚体，所述组合物或所述药用化合物用于哺乳动物特别是人的剂量可以介于0.001－1000mg/kg体重/天，例如介于0.01－100mg/kg体重/天，又例如介于0.1－10mg/kg体重/天。
有益效果
本发明的脂肽LP-19具有广谱、强效和长效的优点：
1、LP-19具有较强的广谱抗HIV-1、HIV-2和SIV活性，尤其对HIV-2和SIV显示很强的优势作用:LP-19对HIV-1、HIV-2和SIV介导的细胞膜融合均具有强效的抑制作用，显著高于三个对照多肽T-20、2P23和LP-11(图3)；LP-19对HIV-1和SIV介导的细胞侵入均具有强效的抑制作用，显著高于三个对照多肽T-20、2P23和LP-11(图4)。LP-19对感染性HIV-1和HIV-2的抑制活性均显著高于三个对照多肽T-20、2P23和LP-11(图5)。
2、LP-19对各种亚型HIV-1病毒的抑制活性也显著高于三个对照多肽T-20、2P23和LP-11(图6)。
3、LP-19抑制T-20耐药株的活性比T-20、2P23、LP-11分别高出10613.53倍、6.18倍和2.18倍；LP-19抑制2P23耐药株的活性比T-20、2P23、LP-11分别高出173.63倍、45.09和6.83倍(图7)。
4、LP-19在猴子体内表现为显著的强效和长效活性，皮下途径抑制峰值为T-20的100倍以上，静脉途径抑制峰值为T-20的2000倍以上。LP-19即使在皮下注射60和72小时，其抑制峰值血清最大稀释倍数分别为66倍和40倍，类似于T-20在2和4小时的表现(图8)。
LP-19的衍生物LP-20至LP-32具有广谱活性，既对HIV-1有效，也对HIV-2和SIV有效(图1)。
附图说明
图1为具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽的结构及其抗病毒 活性。
图2为HIV融合蛋白gp41的结构与功能及多肽膜融合抑制剂。其中，FP指gp41融合肽；NHR指N-末端重复序列；CHR指C-末端重复序列；6-HB指六聚体螺旋。多肽序列中标记为黑体的氨基酸为形成M-T钩子的残基，下划线部分为口袋结合区(PBD)。图2中所有多肽或脂肽的氨基末端均连接乙酰基作为氨基端保护基，所有多肽或脂肽的羧基末端均连接氨基作为羧基端保护基。
图3为LP-19和对照多肽对HIV-1、HIV-2和SIV介导的细胞膜融合的抑制作用。
图4为LP-19和对照多肽对HIV-1和SIV假病毒细胞进入的抑制作用。
图5为LP-19和对照多肽对HIV-1和HIV-2感染的抑制作用。
图6为LP-19和对照多肽对各种亚型HIV-1的抑制作用。
图7为LP-19和对照多肽T-20对2P23耐药毒株的抑制作用。
图8为LP-19和对照多肽注射猕猴血清的抗病毒活性。图中，M248、M249、M250、M252、M253和M254为猴子的编号。
图9为2P23和LP-19自身螺旋结构及其与NHR相互作用的圆二色谱分析。图中，NA表示由于在测量温度范围内螺旋结构没能充分解离而无法准确确定其Tm值(not applicable)。
本发明中氨基酸的缩写具有本领域公知的含义，E为谷氨酸、M为甲硫氨酸、T为苏氨酸、W为色氨酸、K为赖氨酸、V为缬氨酸、L为亮氨酸、I为异亮氨酸、C为半胱氨酸、Q为谷氨酰胺、N为天冬酰胺、Y为酪氨酸、S为丝氨酸、T为苏氨酸、A为丙氨酸；图1-9中，Ac为乙酰基，PEG8为Fmoc-NH-PEG8-CH2CH2COOH，C16为棕榈酸，C18为硬脂酸，NH2为氨基，Chol为胆固醇，Dih为二氢(神经)鞘氨醇，Toc为维生素E。图3-5中，多肽浓度是指脂肽或多肽的浓度。
本发明的最佳实施方式
本发明的具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽中，最佳的实施方式是上述af)和bf)的脂肽。其中，所述具有抑制HIV-1和/或HIV-2和/或SIV活性的多肽为所述P1(序列表中序列1所示的多肽，即图1中的LP-19、LP-20、LP-21、LP-22和LP-23的第1-23位氨基酸残基所示的多肽，该多肽即为2P23)、P3(序列表中序列2所示的多肽，即图1中的LP-31的第1-31位氨基酸残基所示的多肽)、P5(序列表中序列3所示的多肽，即图1中的LP-32的第1-31位氨基酸残基所示的多肽)、P7(序列表中序列4所示的多肽，即图1中的LP-29的第1-34位氨基酸残基所示的多肽)、P9(序列表中序列5所示的多肽，即图1中的LP-25的第1-25位氨基酸残基所示的多肽)、P11(序列表中序列6所示的多肽，即图1中的LP-24的第1-25位氨基酸残基所示的多肽)、P13(序列表中序列7所示的多肽，即图1中的LP-28的第1-31位氨基酸残基所示的多肽)、P15(序列表中序列8所示的多肽，即图1中的LP-30的第1-30位氨基酸残基所示的多肽)、P17(序列表中序列9所示的多肽，即图1中的LP-26 的第1-27位氨基酸残基所示的多肽)和P19(序列表中序列10所示的多肽，即图1中的LP-27的第1-28位氨基酸残基所示的多肽)。所述亲脂性化合物是棕榈酸(又名软脂酸)(C16)、硬脂酸(C18)、胆固醇(cholesterol)、二氢(神经)鞘氨醇(dihydrosphingosine)或维生素E(tocopherol)。所述连接臂是Fmoc-NH-PEG8-CH2CH2COOH。所述保护基为氨基端保护基和羧基端保护基。所述氨基端保护基为乙酰基，所述羧基端保护基为氨基。这些具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽的名称分别为LP-19至LP-32，它们的结构如图1所示，具有广谱活性(既对HIV-1有效，也对HIV-2和SIV有效)且长效。
上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽的设计思想是来源于HIV融合蛋白gp41CHR区的多肽2P23可以与HIV-1、HIV-2以及SIV的靶序列即NHR序列特异性地结合，亲脂性化合物可以与靶细胞膜结合，使多肽在病毒感染部位的浓度得以提高，从而提高多肽的抗病毒活性。同时，亲脂性化合物还具有与血浆蛋白(如HSA)结合从而提高多肽稳定性和生物半衰期的作用。多肽序列与亲脂性化合物通过柔性连接臂聚乙二醇(PEG)连接，以克服空间障碍使脂肽比较灵活地发挥功能(图2)。上述具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽对来源于HIV-1、HIV-2和SIV的NHR靶序列具有较强的结合能力，对病毒都具有极强的抑制作用，在非人灵长类动物(猴子)体内表现出显著的长效活性。
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。下述实施例中的所有多肽的氨基酸均为L-型氨基酸。
本发明的实施方式
实施例1、脂肽的设计
本实施例设计了具有抑制HIV-1和/或HIV-2和/或SIV活性的脂肽，它们的名称分别为LP-19、LP-20、LP-21、LP-22、LP-23、LP-24、LP-25、LP-26、LP-27、LP-28、LP-29、LP-30、LP-31和LP-32。
LP-19、LP-20、LP-21、LP-22、LP-23、LP-24和LP-25的结构如图1所示，其设计理念包括：(1)其多肽序列N端含有M-T钩子结构基序(EMT)，以增强多肽与NHR疏水口袋区的亲和力；(2)其多肽序列与NHR相互作用的氨基酸来源于HIV-1/HIV-2/SIV序列，以增加多肽的广谱抗病毒能力；(3)多肽序列的非NHR结合氨基酸替换成“i和i+3”双离子对结构(即EE--KK结构)，以便通过形成“盐桥”结构促进多肽的螺旋性；(4)在多肽的C端通过一个PEG8连接臂和一个赖氨酸(或半胱氨酸)偶联一个亲脂性化合物，以使多肽具有结合靶细胞膜和/或病毒膜的能力，从而提高病毒入侵局部的多肽浓度；(5)亲脂性化合物修饰多肽具有与血浆蛋白(如HSA)结合的能力，从而提高其生物稳定性。
LP-26、LP-27、LP-28、LP-29、LP-30、LP-31和LP-32的结构如图1所示，除 多肽序列不同和不含连接臂外，其他均与LP-19或LP-20相同。
实施例2、多肽和脂肽的制备
1、多肽的制备
多肽T-20和2P23的氨基末端均连接乙酰基作为氨基端保护基，羧基末端均连接氨基作为羧基端保护基，这些多肽的结构如图2所示。这些多肽的合成均采用标准的固相多肽合成法(Fmoc/tBu策略)，由羧基端向氨基端方向手动合成。所有多肽序列均按照多肽合成的常规将C-端酰胺化，N-端乙酰化。采用N-芴甲氧羰(Fmoc)保护的氨基酸、采用Rink树脂(取代常数为0.44mmol/g)作为固相载体，用25％(体积百分含量)哌啶的DMF溶液脱除氨基保护基Fmoc，每次脱除步骤需进行两次，时长分别为8min和10min。接肽反应选用的缩合方法为DIPC/HOBt法和PyBOP法，氨基酸和活化试剂均采用3倍当量，反应时长为1小时，采用茚三酮定性显色(Kaiser法)监测反应进程。若某个氨基酸缩合反应不完全，适当延长反应时间或重复缩合一次，直至得到所需的目标肽段。使用切割试剂(三氟乙酸∶1,2-乙二硫醇：苯甲硫醚：苯酚：H2O：三异丙基硅烷＝68.5:10:10:5:3.5:1，v/v)将目标多肽从树脂上裂解下来并除去侧链保护基(30摄氏度下切割4小时)。进行过滤操作，将滤液加入到大量冷的无水乙醚中使多肽沉淀析出，离心。用乙醚洗涤数次后干燥，即得到多肽粗品。
2、脂肽的制备
按照如下方法制备结构如图1所示的LP-19、LP-20、LP-21、LP-22、LP-23、LP-24、LP-25、LP-26、LP-27、LP-28、LP-29、LP-30、LP-31和LP-32。
棕榈酸(LP-19、LP-26至LP-30)、硬脂酸(LP-20、LP-24、LP-25、LP-31、LP-32)、二氢(神经)鞘氨醇(LP-22)或维生素E(LP-23)对多肽的修饰是通过多肽C端Lys的侧链氨基进行酰胺化反应完成(参考背景技术中参考文献15：Chong H,Wu X,Su Y,He Y.Development of potent and long-acting HIV-1fusion inhibitors.AIDS 2016,30(8):1187-1196)。胆固醇(LP-21)对多肽的修饰是通过多肽C端Cys侧链的巯基和溴乙酸胆固醇酯进行化学选择性极高的成硫醚反应接枝到多肽链上(参考背景技术中参考文献14：Ingallinella P,Bianchi E,Ladwa NA,Wang YJ,Hrin R,Veneziano M,et al.Addition of a cholesterol group to an HIV-1peptide fusion inhibitor dramatically increases its antiviral potency.Proc Natl Acad Sci U S A 2009,106:5801-5806)。
下面以LP-19为例，阐述脂肽的合成方法：
所用的化学试剂，如Rink Amide MBHA树脂，Fmoc-PEG4-OH(Fmoc-15-amino-4,7,10,13-tetraoxapentadecacanoic acid)，各种Fmoc氨基酸、棕榈酰氯(Palmitoyl chloride)、N,N'-二异丙基碳二亚胺(DIC)、1-羟基苯并三唑(HOBt)、三氟乙酸(TFA)、乙二硫醇(EDT)、茚三酮、六氢吡啶(PIPE)、苯酚、N,N’-二甲基甲酰胺(DMF)、色谱纯乙腈等均从主要化学试剂供应商购买， 使用前未经过进一步的提纯。
采用标准的手动固相Fmoc法，以Rink Amide MBHA树脂(取代常数0.34mmol/g)为起始原料，从C端向N端方向合成。用25％六氢吡啶/DMF(体积比)去除Rink树脂上的Fmoc保护基，然后用2倍当量Fmoc-Lys(Dde)-OH/HOBt/DIC与树脂进行接枝引入C端第一个氨基酸残基。而后，再次用25％六氢吡啶/DMF(体积比)去除N端Fmoc保护基使N端成为自由氨基。如此反复依次连接两个PEG4及各个氨基酸残基。
所用原料及用量分别对应为Fmoc-PEG4-OH(1.5eq)，Fmoc-PEG4-OH(1.5eq)，Fmoc-Lys(Boc)-OH(3eq)，Fmoc-Leu-OH(3eq)，Fmoc-Leu-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Ile-OH(3eq)，Fmoc-Lys(Boc)-OH(3eq)，Fmoc-Lys(Boc)-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Leu-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Val-OH(3eq)，Fmoc-Lys(Boc)-OH(3eq)，Fmoc-Lys(Boc)-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Trp(Boc)-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Glu(OtBu)-OH(3eq)，Fmoc-Trp(Boc)-OH(3eq)，Fmoc-Thr(tBu)-OH(3eq)，Fmoc-Met-OH(3eq)和Fmoc-Glu(OtBu)-OH(3eq)。最后进行N端乙酰化封端(3倍当量Ac2O，6倍当量二异丙基乙胺)，完成主链的合成。其中各步骤反应时间如下：去保护8分钟，两次；接枝普通氨基酸60分钟，接枝PEG4时间为180分钟。
用2％水合肼/DMF溶液(体积比)处理树脂，以去除C端Lys的侧链的保护基，而后用3倍当量棕榈酰氯与6倍当量二异丙基乙胺混合后与C端Lys的侧链氨基进行酰胺化反应(60分钟)，从而实现C端Lys残基上的棕榈酰化修饰。
以上每步反应后都需用DMF洗涤树脂六次以上，并且都通过茚三酮定性显色(Kaiser法)监测反应进程，若某个氨基酸缩合反应不完全，重复缩合一次，直至得到所需的目标肽段。
切割及去侧链保护：脂肽合成完毕后，真空干燥树脂。往干燥后的树脂中加入切割试剂(三氟乙酸：1,2-乙二硫醇：苯甲硫醚：苯酚：H2O：三异丙基硅烷＝68.5:10:10:5:3.5:1,v/v)，切割条件为30摄氏度下切割3小时，将目标脂肽从树脂上裂解下来并除去侧链保护基。进行过滤操作，将滤液加入到大量冷的无水乙醚中使脂肽沉淀析出，离心。用乙醚洗涤数次后干燥，即得到脂肽粗品。
3、多肽和脂肽的纯化
多肽和脂肽的纯化及表征：多肽粗品和脂肽粗品的纯化在反相高效液相色谱仪上进行。所用色谱柱柱料为10微米粒径的反相C18硅胶，孔径为100埃，色谱柱尺寸为50×250mm。
色谱操作条件：线性梯度洗脱，洗脱液由流动相A和流动相B组成。流动相A为三氟乙酸和乙腈的水溶液，三氟乙酸的体积百分浓度为0.05％，乙腈的体积百分浓度为2％。流动相B为90％(体积百分浓度)乙腈水溶液。线性梯度洗脱由20％B到40％B，时间20分钟，洗脱流速为每分钟25毫升，紫外检测波长 220纳米。冻干溶剂后得到蓬松状态的脂肽纯品，其化学结构由MALDI-TOF质谱进行表征，而其纯度则由分析型高效液相色谱仪(流速：每分钟1毫升)给出。其中，分析型高效液相色谱仪的型号：岛津CBM-10A VP PULS，所采用的色谱柱的型号:Agela4.6*250mm C18。色谱操作条件：线性梯度洗脱，洗脱液由流动相A和流动相B组成。流动相A为三氟乙酸和乙腈的水溶液，三氟乙酸的体积百分浓度为0.05％，乙腈的体积百分浓度为2％。流动相B为三氟乙酸和乙腈的水溶液，三氟乙酸的体积百分浓度0.05％，乙腈的体积百分浓度为90％。线性梯度洗脱由25％B到45％B，时间20分钟。得到的多肽和脂肽纯品由分析型反相高效液相色谱仪表征表明LP-19、LP-20、LP-21、LP-22、LP-23、LP-24、LP-25、LP-26、LP-27、LP-28、LP-29、LP-30、LP-31LP-32、T-20和2P23的纯度均大于95％。
实施例3、脂肽的广谱抗病毒活性检测
3.1实验材料与方法
3.1.1脂肽对HIV和SIV介导的细胞融合的抑制作用
HIV-1、HIV-2和SIV介导的细胞融合抑制实验的材料和方法参照Xiong S等发表的文献(背景技术中参考文献13)。其中，HEK293T细胞(简称239T细胞)购自美国模式培养物集存库(ATCC)；U87CD4+CXCR4+细胞由美国NIH艾滋病试剂和参照物项目提供(目录号：4036)；HIV-2毒株ROD分子克隆的质粒pROD由葡萄牙里斯本大学Nuno Taveira教授惠赠；表达SIV毒株SIVpbj和SIV239包膜蛋白的质粒(分别为pSIVpbj-Env和pSIV239)由复旦大学徐建青教授惠赠；荧光报告系统质粒DSP1-7和DSP8-11由日本东京大学Zene Matsuda教授惠赠；表达HIV-1毒株NL4-3包膜蛋白的质粒是将HIV-1毒株NL4-3的包膜蛋白(ENV)编码基因插入载体pcDNA3.1(-)得到的重组表达质粒。首先将239T细胞(效应细胞)加入到96孔细胞培养板中(1.5×104个/孔)，将U87CD4+CXCR4+(靶细胞)加入到24孔细胞培养板中(8×104个/孔)，两者分别于37摄氏度、5％CO2的条件下培养过夜。第二天，将表达毒株包膜蛋白(Env)的质粒和DSP1-7质粒按1：1混合并转染239T细胞，将DSP8-11质粒转染U87CD4+CXCR4+细胞。于37摄氏度、5％CO2的条件下培养48小时后，将DSP8-11质粒转染的U87CD4+CXCR4+细胞重悬于300微升预温的培养液，并加入0.05微升EnduRen活细胞底物(Promega)。将待测物T-20、2P23、LP-11或LP-19用二甲基亚砜(DMSO)溶解并用细胞培养液稀释后，按3倍倍比稀释加入到96孔培养板的效应细胞孔，然后转移75微升靶细胞于效应细胞。轻轻离心以使效应细胞和靶细胞充分接触后，放置于37摄氏度培养1小时，并测定荧光素酶的活性(相对荧光单位，RLU)。计算每一浓度样品抑制率，利用GraphPad Prism Software 2.01软件计算半数有效抑制剂量(IC50值)。
3.1.2对HIV-1和SIV假病毒的抑制作用
采用HIV重组假病毒系统评价多肽对病毒进入靶细胞的抑制作用，方法参照 Xiong S等发表的文献[背景技术参考文献13],表达HIV-1或SIV包膜蛋白的质粒同上面3.1.1所述。基本步骤包括(1)假病毒的制备：将表达HIV-1NL4-3、SIVpbj或SIV239毒株包膜蛋白的质粒和HIV骨架质粒pSG3△ENV(表达HIV基因组中除ENV之外的所有蛋白，由美国NIH艾滋病试剂和参照物项目提供，目录号为11051)，按质量比1:2的比例转染293T细胞，同时设只转染相同量的pSG3△ENV的对照。于37摄氏度、5％CO2细胞培养箱中孵育6小时后换液，然后继续孵育48小时使假病毒分泌至上清中。用移液器尽量多地吸出细胞培养瓶或细胞培养板中的上清，经0.45μm滤器过滤或1000g离心10分钟收取上清，向其中加入胎牛血清(FBS)使其终浓度为20％，转移至聚丙烯管中于-80摄氏度保存备用或直接进行病毒滴定。(2)HIV假病毒的滴定：将病毒在96孔板中做5倍稀释，设置4个复孔8个梯度，终体积为100微升。将TZM-bl细胞用胰酶消化并计数，用DMEM完全培养基将细胞稀释至1×105个/ml，每孔加100微升细胞(含15μg/ml DEAE-dextran)，于37摄氏度、5％CO2培养48小时。然后从细胞培养箱中取出96孔板，从上样孔中吸弃上清，加入30微升细胞裂解液，放置10分钟后加入100微升荧光素酶检测试剂。用移液器从每孔中吸出100微升液体，加于对应的96孔白板中，于微孔板光度计读取发光值。用Reed-Muench法计算病毒滴度。(3)抗病毒活性检测：将待测物(T-20、2P23、LP-11或LP-19(用DMSO溶解并用细胞培养液稀释))按倍比(3倍)稀释铺入96孔板中，终体积为50微升，其中用50微升DMEM培养基替代待测物作为阴性对照。加入100微升浓度为1×105个/ml的TZM-bl靶细胞(含15μg/ml DEAE-dextran)，加入上述获得的HIV假病毒50微升(每孔相当于100TCID50)，于37摄氏度、5％CO2条件下培养48小时后，利用荧光素酶检测试剂(Promega)测定每孔的相对荧光单位(RLU)。计算％抑制率和IC50值。
3.1.3对感染性HIV-1(NL4-3)和HIV-2(ROD)毒株的抑制作用
对HIV-1(NL4-3)和HIV-2(ROD)毒株复制的抑制实验参考Xiong S等发表的文献[背景技术参考文献13]。编码HIV-1病毒株NL4-3的分子克隆质粒pNL4-3由美国NIH艾滋病试剂和参照物项目提供(目录号：114)；HIV-2毒株ROD的分子克隆质粒pROD同上面3.1.1所述。采用QIAGEN公司的质粒提取试剂盒制备质粒，采用Invitrogen公司的LipofectamineTM2000转染试剂将质粒转染293T细胞，于37摄氏度、5％CO2细胞培养箱中孵育6小时后换液，然后继续培养48小时。用移液器轻轻收集细胞培养瓶或细胞培养板中的上清，经0.45微米滤器过滤取上清，加入20％FBS，然后分装于聚丙烯管中，放置-80摄氏度保存备用或直接进行病毒滴定，方法同上述HIV假病毒。为测定抗病毒活性，将待测物(T-20、2P23、LP-11或LP-19(用DMSO溶解并用细胞培养液稀释))按倍比(3倍)稀释铺入96孔板中，终体积为50微升，其中用50微升DMEM培养基替代待测物作为阴性对照。加入100微升TZM-bl细胞(105个细胞/ml，含15μg/ml DEAE-dextran)，加入已获得的病毒50微升，每孔相当于100TCID50。培养48小时后，利用荧光素酶检测试剂(Promega)测定每孔的相对荧光单位(RLU)。计算％抑制率和IC50值。
3.2实验结果及分析
3.2.1 LP-19对HIV-1、HIV-2和SIV介导的细胞融合的抑制作用
首先，分析了LP-19对各种病毒介导的细胞融合的抑制活性，并以T-20、2P23和LP-11作为对照。结果如图3所示，LP-19对HIV-1、HIV-2和SIV介导的细胞膜融合均具有强效的抑制作用，显著高于三个对照多肽。LP-19对HIV-1毒株NL4-3介导的细胞融合的抑制IC50为0.14nM,而T-20、2P23和LP-11对其的抑制IC50值分别为7.17nM、0.28nM和0.78nM；LP-19对HIV-2毒株ROD介导的细胞融合的抑制IC50为2.27nM,而T-20、2P23和LP-11对其的抑制IC50值分别为569.8nM、12.25nM和20.96nM；LP-19对SIVpbj介导的细胞融合的抑制IC50为0.67nM,而T-20、2P23和LP-11对其的抑制IC50值分别为5.44nM、1.91nM和4.18nM；LP-19对SIV293介导的细胞融合的抑制IC50为2.28nM,而T-20、2P23和LP-11对其的抑制IC50值分别为219nM、2.64nM和11.91nM。
3.2.2 LP-19对HIV-1和SIV假病毒的抑制作用
进一步对LP-19及对照多肽对假病毒介导的细胞侵入的抑制作用进行了评价，结果如图4所示。T-20、2P23、LP-11和LP-19对HIV-1NL4-3介导的细胞侵入的抑制IC50分别为78.78nM、0.78nM、0.21nM和0.12nM，对SIVpbj介导的细胞侵入的抑制IC50分别为246.41nM、10.56nM、17.38nM和0.48nM，对SIV239介导的细胞侵入的抑制IC50分别为402.77nM、3.67nM、2.9nM和0.58nM。LP-19对HIV-1和SIV介导的细胞侵入均具有强效的抑制作用，显著高于T-20、2P23和LP-11三个对照多肽。
3.2.3 LP-19对HIV-1和HIV-2感染的抑制作用
同时，检测了对感染性HIV-1和HIV-2的抑制活性，结果如图5所示。T-20、2P23、LP-11和LP-19对HIV-1毒株NL4-3的抑制IC50分别为112.75nM、0.62nM、0.22nM和0.16nM，对HIV-2毒株ROD的抑制IC50分别为353.68nM、16.21nM、8.09nM和1.66nM。LP-19对感染性HIV-1和HIV-2的抑制活性均显著高于三个对照多肽T-20、2P23和LP-11。结合3.2.1、3.2.2和3.2.3的实验结果可以看出，LP-19具有较强的广谱抗HIV-1、HIV-2和SIV活性，尤其对HIV-2和SIV显示很强的优势作用。
3.2.4 LP-19对各种亚型HIV-1的抑制作用
世界范围内艾滋病主要由HIV-1引起,由于病毒的变异产生了多种亚型,包括A-D、F-H、J和K亚型等。其中A，B和C亚型是引起世界艾滋病流行的主要病毒。而在中国，B/C和A/E重组病毒的流行占据主导地位。为进一步评价抗LP-19的活性，制备了一组29株HIV-1假病毒，包括国际代表毒株和中国目前流行的HIV毒株，其中有A亚型3株、B亚型6株、B’亚型3株、C亚型6株、G亚型1株、A/C重组型1株、A/E重组型4株、和B/C重组型5株。这些假病毒毒株由中国医学科学院病原生物学研究所何玉先教授实验室保存，参见背景技术参考文献13。参照上述3.1.2中的方法(对HIV-1和SIV假病毒的抑制作用)测定T-20、2P23、LP-11 和LP-19的抗病毒活性。抗病毒实验结果表明，T-20、2P23、LP-11和LP-19抑制各种亚型HIV-1毒株的平均IC50分别为34.01nM、5.22nM、1.29nM和0.47nM。可见，LP-19对各种亚型HIV-1病毒的抑制活性也显著高于T-20、2P23和LP-11三个对照多肽(图6)。
3.2.5 LP-19对T-20和2P23耐药病毒株的抑制作用
T-20是目前唯一批准用于临床治疗的HIV膜融合抑制剂，然而其活性不但明显低于新一代的多肽，而且很容易诱导耐药突变，导致临床抗病毒治疗的失败。2P23是发明人新设计仅有23个氨基酸的含有M-T钩子结构的螺旋短肽，主要靶向融合蛋白gp41的NHR疏水口袋结构，对HIV-1、HIV-2和SIV均具有较好的抑制活性，但对一些短肽诱导的耐药突变位点也呈现一定的交叉耐药性。为更充分评价LP-19的抗病毒广谱性和优势，发明人制备了携带NHR突变的假病毒，分别含有T-20和2P23对应的耐药位点(图7)。这些制备假病毒的质粒由发明人基于表达HIV-1毒株NL4-3包膜蛋白的质粒pNL4-3-Env通过定点突变获得，由中国医学科学院病原生物学研究所何玉先教授实验室保存并常规使用。图7中的T-20耐药毒株分别为(Chong H,Yao X,Zhang C,Cai L,Cui S,Wang Y,He Y.Biophysical Property and Broad Anti-HIV Activity of Albuvirtide,a 3-Maleimimidopropionic Acid-Modified Peptide Fusion Inhibitor.PLoS One.2012,7(3):e32599.)的表3中的HIV-1NL4-3突变毒株，本发明图7中毒株名称的下标即为该文献中表3中的毒株名称。2P23耐药毒株分别为(Su Y,Chong H,Xiong S,Qiao Y,Qiu Z，He Y.Genetic pathway of HIV-1resistance to novel fusion inhibitors targeting the Gp41pocket.J Virol.2015，89(24):12467-12479)中的表1中HIV-1NL4-3突变毒株，本发明图7中毒株名称的下标即为该文献中表1中的毒株名称。参照上述3.1.2中的方法测定T-20、2P23、LP-11和LP-19的抗病毒活性。抗病毒实验结果表明，T-20、2P23、LP-11和LP-19抑制T-20耐药毒株的平均IC50分别为1804.3nM、1.05nM、0.37nM和0.17nM(图7)。可见，LP-19抑制T-20耐药株的活性比T-20、2P23、LP-11分别高出10613.53倍、6.18倍和2.18倍；LP-19抑制2P23耐药毒株的平均IC50分别为134.4nM、36.52nM、5.53nM和0.81nM。可见，LP-19抑制2P23耐药株的活性比T-20、2P23、LP-11分别高出165.93倍、45.09和6.83倍。
实施例4、LP-19的体内抗病毒活性检测
上述多个基于细胞水平的体外抗病毒实验的结果表明，LP-19是一个广谱的高活性的抗病毒脂肽。为明确LP-19是否在体内也具有很强的抗病毒活性，将LP-19以及对照多肽T-20和2P23通过皮下或静脉注射到猴子体内，然后采取不同时间点的血液标本在体外测定血清的抗病毒活性。该方法不但可以了解待测物的体内抗病毒活性，也有助于分析待测物在体内的稳定性和半衰期。具体方法如下：选择6只实验猕猴(恒河猴)，雌雄各半，年龄3-4岁、体重3.4-4.7kg。按每公斤体重3毫克(3mg/Kg)皮下注射待测物T-20、2P23或LP-19(均用无菌蒸馏水溶解)，分 别于注射前(0小时)，注射后1、2、4、6、8、12、18、24、36、48、60、72小时抽取0.4ml静脉血标本，并按常规方法分离血清。LP-19除皮下途径注射外，另外采用静脉途径注射LP-19(用无菌蒸馏水溶解)，注射剂量为3mg/Kg体重。每次实验间隔时间2周以上，以确保没有上次注射待测物的残留。按照实施例3中的方法采用抗病毒实验检测血清抑制HIV-1假病毒NL4-3突变株D36G(参见背景技术中文献11的表2)的活性。血清3倍倍比稀释。实验结果如图8所示，皮下注射T-20在2和4小时表现为抑制峰值，抑制50％NL4-3感染性的血清最大稀释倍数分别为45倍和46倍；皮下注射短肽2P23在1和2小时表现为抑制峰值，血清最大稀释倍数分别为60倍和68倍；皮下注射短肽LP-19在6和8小时表现为抑制峰值，血清最大稀释倍数分别为5396倍和4720倍；静脉注射LP-19在1和2小时即表现为抑制峰值，血清最大稀释倍数分别为99107倍和76346倍。可见，LP-19在猴子体内表现为显著的强效和长效活性，皮下途径抑制峰值为T-20的100倍以上，静脉途径抑制峰值为T-20的2000倍以上。LP-19即使在皮下注射60和72小时，其抑制峰值血清最大稀释倍数分别为66倍和40倍，类似于T-20在2和4小时的表现。
实施例5. LP-19的结构及其与NHR靶序列的相互作用分析
5.1实验材料与方法
采用圆二色谱技术(CD)测定多肽的二级结构和螺旋稳定性(Tm值)，实验方法参考文献[14]。圆二色谱仪为日产Jasco-815。首先测定LP-19及其模板2P23在不同浓度下(分别为20、40、80、160和320μM)的自身螺旋含量和Tm值。然后测定LP-19或2P23与NHR多肽N36(Ac-SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH2)所形成6-HB的螺旋含量和Tm值。2P23、LP-19和N36分别溶于磷酸盐缓冲液(PBS，pH 7.2)中，根据280nm下紫外吸收确定浓度。根据CD信号判断多肽间的相互作用情况及螺旋含量。通过CD温度扫描测定多肽抑制剂与N36形成的六螺旋结构的稳定性。
5.2实验结果及分析
CD测试结果见图9。首先，作为对照的未修饰多肽2P23含有典型的α-螺旋结构(图9中A)，其在20μM、40μM、80μM、160μM和320μM浓度下的螺旋含量分别为76.4％、81.4％、83.8％、84％和79.7％；其在20μM、40μM、80μM和160μM浓度下的Tm值分别为48.1摄氏度、54.7摄氏度、59.9摄氏度和67.8摄氏度(图9中B)。当浓度达到320μM，其螺旋在测定的最高温度(98摄氏度)不能充分解旋而导致Tm无法准确判定，提示其螺旋的高度热稳定性。脂肽LP-19在20μM、40μM、80μM、160μM和320μM浓度下的螺旋含量分别为70％、71.1％、71.9％、76.2％和71％(图9中C)；然而，在测定的最高温度(98摄氏度)，LP-19在各种浓度CD条件下其螺旋都无法充分解旋，说明棕榈酸修饰能够显著增加多肽的螺旋稳定性(图9中D)。进一步比较分析了LP-19和2P23与N36的结合能力。图9中E表明两者都能与N36形成典型的六聚体螺旋结构(6-HB)；图9中F表明2P23/N36复合物的Tm值为79.6摄氏度，但LP-19/N36复合物更加稳定，在测定的最高温度(98 摄氏度)不能充分解旋。从这些CD结果说明，2P23自身为一螺旋多聚体结构，而棕榈酸修饰进一步增加多肽的螺旋稳定性。
实施例6. LP-19衍生脂肽的抗病毒活性
从上述实施例的结果可以看出，LP-19脂肽具有广谱、强效和长效的优点。进一步以其他亲脂化合物取代LP-19中的棕榈酸合成一组新的脂肽(图1)。其中，LP-20、LP-24和LP-25以硬脂酸(C18)修饰，LP-21以胆固醇(Chol)修饰，LP-22以二氢(神经)鞘氨醇(Dih)修饰，LP-23以维生素E(Toc)修饰。同时，还合成了不含PEG8但代之以不同长度对应氨基酸序列的脂肽(LP-26到LP-32)。参照实施例3中3.1.2和3.1.3的抗病毒实验检测了它们对HIV-1NL4-3毒株假病毒、感染性HIV-2ROD毒株和SIV 239毒株假病毒的抑制活性。制备病毒的质粒同上所述。结果如图1所示，这些脂肽都有极强的抗病毒活性。它们既对HIV-1有效，也对HIV-2和SIV有效。
工业实用性
本发明所提供的脂肽、其衍生物、或其可药用盐，所述多聚体，所述组合物或所述药用化合物，可以用于HIV(HIV-1和/或HIV-2)和/或SIV感染的治疗和/或预防。在实际应用中，可以将本发明的脂肽、其衍生物、或其可药用盐，所述多聚体，所述组合物或所述药用化合物作为药物直接给予病人、或者与适宜的载体或赋形剂混合后给予病人，以达到治疗和/或HIV感染的目的。
序列表自由内容
Figure PCTCN2017072885-appb-000001
Figure PCTCN2017072885-appb-000002
Figure PCTCN2017072885-appb-000003
Figure PCTCN2017072885-appb-000004
Figure PCTCN2017072885-appb-000005

Claims (16)

  1. 脂肽、其药用盐、或其衍生物，其特征在于：所述脂肽为下述a)或b)：
    a)所述脂肽包括具有抗病毒活性的多肽，与所述多肽的羧基末端相连的连接臂，与所述连接臂相连的氨基酸X残基和与所述氨基酸X残基相连的亲脂性化合物；所述氨基酸X为K、C、S、T或Y；
    b)所述脂肽包括具有抗病毒活性的多肽，与所述多肽的羧基末端相连的亲脂性化合物；
    所述a)或b)中，所述病毒为下述v1-v7中的任一：
    v1、HIV-1、HIV-2和SIV；
    v2、HIV-1和HIV-2；
    v3、HIV-1和SIV；
    v4、HIV-2和SIV；
    v5、HIV-1；
    v6、HIV-2；
    v7、SIV。
  2. 根据权利要求1所述的脂肽、其药用盐、或其衍生物，其特征在于：所述连接臂为Fmoc-NH-PEGn-CH2CH2COOH，n为2、3、4、6、8、10、11或12；和/或，
    所述a)或b)中，所述亲脂性化合物为含8到20个碳原子的脂肪酸或胆固醇、二氢鞘氨醇或维生素E。
  3. 根据权利要求2所述的脂肽、其药用盐、或其衍生物，其特征在于：所述含8到20个碳原子的脂肪酸为棕榈酸或硬脂酸。
  4. 根据权利要求3所述的脂肽、其药用盐、或其衍生物，其特征在于：所述a)或b)中，所述多肽为P1-P20中的任一种：
    P1、序列表中序列1所示的多肽
    P2、在序列表中序列1所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P3、序列表中序列2所示的多肽；
    P4、在序列表中序列2所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P5、序列表中序列3所示的多肽；
    P6、在序列表中序列3所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P7、序列表中序列4所示的多肽；
    P8、在序列表中序列4所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P9、序列表中序列5所示的多肽；
    P10、在序列表中序列5所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P11、序列表中序列6所示的多肽；
    P12、在序列表中序列6所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P13、序列表中序列7所示的多肽；
    P14、在序列表中序列7所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P15、序列表中序列8所示的多肽；
    P16、在序列表中序列8所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P17、序列表中序列9所示的多肽；
    P18、在序列表中序列9所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P19、序列表中序列10所示的多肽；
    P20、在序列表中序列10所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性。
  5. 根据权利要求4所述的脂肽、其药用盐、或其衍生物，其特征在于：所述衍生多肽由20到34个氨基酸残基组成。
  6. 根据权利要求5所述的脂肽、其药用盐、或其衍生物，其特征在于：所述a)或b)中，所述脂肽还包括氨基端保护基和/或羧基端保护基，所述氨基端保护基连接在所述多肽的氨基末端上，所述羧基端保护基连接在所述脂肽的所述羧基末端上。
  7. 根据权利要求6所述的脂肽、其药用盐、或其衍生物，其特征在于：所述脂肽为af)或bf)：
    af)、由所述具有抗病毒活性的多肽，与所述多肽的羧基末端相连的连接臂，与所述连接臂相连的氨基酸X残基和与所述氨基酸X残基相连的亲脂性化合物和保护基组成；所述氨基酸X为K、C、S、T或Y；所述保护基为氨基端保护基和/或羧基端保护基；
    bf)、由所述具有抗病毒活性的多肽，与所述多肽的羧基末端相连的亲脂性化合物和保护基组成；所述保护基为氨基端保护基和/或羧基端保护基。
  8. 根据权利要求7所述的脂肽、其药用盐、或其衍生物，其特征在于：所述连接臂为Fmoc-NH-PEG8-CH2CH2COOH。
  9. 多肽、其药用盐、或其衍生物，其特征在于：所述多肽为P1-P20中的任一种：
    P1、序列表中序列1所示的多肽；
    P2、在序列表中序列1所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P3、序列表中序列2所示的多肽；
    P4、在序列表中序列2所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P5、序列表中序列3所示的多肽；
    P6、在序列表中序列3所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P7、序列表中序列4所示的多肽；
    P8、在序列表中序列4所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P9、序列表中序列5所示的多肽；
    P10、在序列表中序列5所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P11、序列表中序列6所示的多肽；
    P12、在序列表中序列6所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P13、序列表中序列7所示的多肽；
    P14、在序列表中序列7所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P15、序列表中序列8所示的多肽；
    P16、在序列表中序列8所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P17、序列表中序列9所示的多肽；
    P18、在序列表中序列9所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性；
    P19、序列表中序列10所示的多肽；
    P20、在序列表中序列10所示多肽的任何位点添加或替代1个以上氨基酸残基得到的衍生多肽，所述衍生多肽具有所述抗病毒活性。
  10. 根据权利要求9所述的多肽、其药用盐、或其衍生物，其特征在于：所述的多肽衍生物为下述1)-5)中的至少一种：
    1)所述多肽的氨基端连接氨基端保护基和/或所述多肽的羧基端连接羧基端保护基得到的连接物；
    2)所述多肽的羧基端连接寡肽或亲脂性化合物得到的连接物；
    3)所述多肽的氨基端连接寡肽或亲脂性化合物得到的连接物；
    4)所述多肽的氨基端和羧基端均连接寡肽或亲脂性化合物得到的连接物；
    5)所述多肽被蛋白质、聚乙二醇、马来酰亚胺修饰得到的修饰物。
  11. PM1或PM2的多聚体：
    PM1、由权利要求1所述的脂肽、其药用盐、或其衍生物形成的多聚体；
    PM2、由权利要求9所述的多肽、其药用盐、或其衍生物形成的多聚体。
  12. 一种组合物，其包含C1)和C2)：C1)为C11)、C12)或/和C13)；所述C11)为权利要求1所述的脂肽、其衍生物、或其可药用盐；所述C12)为权利要求9所述的多肽、其衍生物、或其可药用盐；所述C13)为权利要求11所述的多聚体；
    C2)药学上可接受的载体或辅料；
    所述组合物具有下述F1)-F5)中的至少一种功能：
    F1)抗病毒；
    F2)治疗和/或预防和/或辅助治疗病毒感染所致疾病；
    F3)抑制病毒进行细胞融合；
    F4)抑制病毒侵入细胞；
    F5)抑制病毒复制；
    F1)-F5)中,所述病毒为下述v1-v7中的任一：
    v1、HIV-1、HIV-2和SIV；
    v2、HIV-1和HIV-2；
    v3、HIV-1和SIV；
    v4、HIV-2和SIV；
    v5、HIV-1；
    v6、HIV-2；
    v7、SIV。
  13. C11)、C12)、C13)或/和C14)在制备E1)-E5)中至少一种产品中的应用：
    所述C11)为权利要求1所述的脂肽、其衍生物、或其可药用盐；所述C12)为权利要求9所述的多肽、其衍生物、或其可药用盐；所述C13)为权利要求11所述的多聚体；所述C14)为权利要求12所述的组合物；
    所述E1)为抗病毒的产品；
    所述E2)为治疗和/或预防和/或辅助治疗病毒感染所致疾病的产品；
    所述E3)为抑制病毒进行细胞融合的产品；
    所述E4)为抑制病毒侵入细胞的产品；
    所述E5)为抑制病毒复制的产品；
    所述E1)-E5)中，所述病毒为下述v1-v7中的任一：
    v1、HIV-1、HIV-2和SIV；
    v2、HIV-1和HIV-2；
    v3、HIV-1和SIV；
    v4、HIV-2和SIV；
    v5、HIV-1；
    v6、HIV-2；
    v7、SIV。
  14. 药用化合物，其特征在于：所述药用化合物为C11)、C12)或C13)；所述C11)为权利要求1所述的脂肽、其衍生物、或其可药用盐；所述C12)为权利要求9所述的多肽、其衍生物、或其可药用盐；所述C13)为权利要求11所述的多聚体。
  15. 根据权利要求14所述的药用化合物，其特征在于：所述药用化合物具有下述F1)-F5)中的至少一种功能：
    F1)抗病毒；
    F2)治疗和/或预防和/或辅助治疗病毒感染所致疾病；
    F3)抑制病毒进行细胞融合；
    F4)抑制病毒侵入细胞；
    F5)抑制病毒复制；
    F1)-F5)中,所述病毒为下述v1-v7中的任一：
    v1、HIV-1、HIV-2和SIV；
    v2、HIV-1和HIV-2；
    v3、HIV-1和SIV；
    v4、HIV-2和SIV；
    v5、HIV-1；
    v6、HIV-2；
    v7、SIV。
  16. 治疗或/和预防病毒感染动物的方法，包括给受体动物施用C11)、C12)、C13)或/和C14)以抑制病毒感染动物；
    所述C11)为权利要求1所述的脂肽、其衍生物、或其可药用盐；所述C12)为权利要求9所述的多肽、其衍生物、或其可药用盐；所述C13)为权利要求11所述的多聚体；所述C14)为权利要求12所述的组合物；
    所述病毒为下述v1-v7中的任一：
    v1、HIV-1、HIV-2和SIV；
    v2、HIV-1和HIV-2；
    v3、HIV-1和SIV；
    v4、HIV-2和SIV；
    v5、HIV-1；
    v6、HIV-2；
    v7、SIV。
PCT/CN2017/072885 2017-02-04 2017-02-04 广谱抑制hiv的脂肽、其衍生物、其药物组合物及其用途 WO2018141089A1 (zh)

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